VIRAL VECTORS FOR THE TREATMENT OF RETINAL DYSTROPHY

Abstract
The present invention relates to viral vectors that are capable of delivering a heterologous gene to the retina and in particular delivering RLBP1 to RPE and Müller cells of the retina. The invention also relates nucleic acids useful for producing viral vectors, compositions comprising the viral vectors and uses of the compositions and viral vectors. The invention also relates to methods of delivering and/or expressing a heterologous gene to the retina, improving the rate of dark adaption in a subject and treating RLBP1-associated retinal dystrophy.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 16, 2013 is named PAT055109-US-NP_SL.txt and is 253,289 bytes in size.


BACKGROUND OF THE INVENTION

Retinitis pigmentosa (RP) refers to a group of inherited degenerations of the photoreceptor cells (rods and cones) of the retina leading to visual loss and blindness. Mutations in any of a wide variety of genes can cause RP, including genes encoding proteins that are involved in phototransduction (the process by which the energy of a photon of light is converted in the photoreceptor cell outer segment into a neuronal signal), the visual cycle (production and recycling of vitamin A in the retina), photoreceptor structure, and transcription factors (Phelan and Bok, 2000).


RLBP1-associated retinal dystrophy is a rare form of RP caused by mutations in the retinaldehyde binding protein 1 (RLBP1) gene on chromosome 15. Mutations in this gene cause absence of or dysfunction of cellular retinaldehyde-binding protein (CRALBP), a protein that is important in the visual cycle (He et al 2009). CRALBP is expressed in retinal pigment epithelium (RPE) and Müller cells, ciliary epithelium, iris, cornea, pineal gland and a subset of oligodendrocytes of the optic nerve and brain (Saari et al 1997). CRALBP accepts 11-cis-retinol from the isomerase RPE65 and acts as a carrier of this substrate for 11-cis-retinol dehydrogenase (RDH5) to convert the substrate into 11-cis-retinal. The rate of chromophore regeneration is severely reduced in the absence of functional CRALBP (Travis et al 2007). The function of CRALBP outside the RPE is not well understood, but it has been suggested that CRALBP in the Müller cells supports a cone-specific visual pathway that permits cone cells to quickly adapt to a wide range of light intensities (Wang and Kefalov 2011).


RLBP1-associated retinal dystrophy is characterized by early severe night blindness and slow dark adaptation, followed by progressive loss of visual acuity, visual fields and color vision leading to legal blindness typically around middle adulthood. The fundus appearance is characterized by yellow or white spots in the retina. The reduction in visual acuity and visual field significantly impacts patients' quality of life (Burstedt and Mönestam, 2010).


The most common RLBP1 mutations leading to RLBP1-associated retinal dystrophy are recessive mutations, designated R234W and M226K (Golovleva I and Burstedt M 2012). RLBP1-associated retinal dystrophy caused by 1 or both of these recessive missense mutations is also known as Bothnia Dystrophy. Several other loss-of-function mutations in the RLBP1 gene have been reported to lead to RLBP1-associated retinal dystrophy. For example, splice-junction mutations in RLBP1 cause rod-cone dystrophy in Newfoundland. Currently there is no treatment available for RLBP1-associated retinal dystrophy (Eichers et al 2002).


The present invention is based in part on the discovery that expression of RLBP1 from recombinant adeno-associated viral vectors (rAAV) having a combination of selected promoter, AAV genome and capsid serotype provides a potent and efficacious treatment for RLBP1-associated retinal dystrophy.


SUMMARY OF THE INVENTION

The present invention relates generally to recombinant viral vectors and methods of using recombinant viral vectors to express proteins in the retina of subjects suffering from retinal diseases and blindness.


The present invention relates to viral vectors that are capable of delivering a heterologous gene to the retina. The present invention also relates to viral vectors that are capable of directing a heterologous gene to RPE and Müller cells of the retina. The present invention further relates to viral vectors that are recombinant adeno-associated viral vectors (rAAV). In certain embodiments the rAAV viral vector may be selected from among any AAV serotype known in the art, including, without limitation, AAV1-AAV12. In certain embodiments, the rAAV vector capsid is an AAV2 serotype. In certain other embodiments, the rAAV vector capsid is an AAV8 serotype.


The invention relates, in part, to viral vectors carrying a single stranded vector genome. In the single stranded viral vector, the vector genome can include a 5′ ITR, a recombinant nucleotide sequence comprising an RLBP1 coding sequence, and a 3′ ITR. The recombinant nucleic acid sequence of the vector genome can also include a promoter as described herein. In one aspect, the promoter is an RLBP1 (long) promoter (SEQ ID NO: 10), in another aspect the promoter is an RLBP1 (short) promoter (SEQ ID NO: 3). In certain specific aspects of the invention, the vector genome comprises, in the 5′ to 3′ direction, nucleic acid sequences selected from: a) SEQ ID NO: 2, 10, 5, 6, 8, and 9; b) SEQ ID NO: 2, 11, 5, 6, 8, 14, 9; c) SEQ ID NO: 2, 22, 5, 6, 8, 23, and 9; and d) SEQ ID NO: 2, 3, 4, 5, 6, 8, 23, and 9.


The invention also relates, in part, to viral vectors carrying a self-complementary genome. The self-complementary vector genome can include, from 5′ to 3′, a 5′ ITR, a first recombinant nucleotide sequence, a non-resolvable ITR (e.g.: ΔITR), a second recombinant nucleotide sequence, and a 3′ ITR, wherein the first and second recombinant nucleotide sequences are self-complementary. The second recombinant nucleotide sequence comprises in the 5′ to 3′ direction, a promoter, an RLBP1 coding sequence and an SV40 polyA sequence. The promoter can be an RLBP1 promoter and, further, can be the RLBP1 (short) promoter (SEQ ID NO: 3). In certain aspects of the invention, the second recombinant nucleotide sequence comprises nucleic acid sequences in the 5′ to 3′ direction of SEQ ID NO: 3, 4, 5, 6, and 8 and the first recombinant nucleotide sequence comprises sequences that are self-complementary to, or the reverse complement of, the second recombinant sequence, for example, SEQ ID NOs: 62, 63, 64, 65, and 66. The invention also relates to a viral vector comprising a self-complementary vector genome wherein the genome comprises, nucleic acid sequences in the 5′ to 3′ direction of: SEQ ID NOs: 36, 62, 63, 64, 65, 66, 1, 3, 4, 5, 6, 8, and 9. The self-complementary vector genome described above can be packaged in an AAV capsid that is selected from any AAV serotype known in the art, including but not limited to AAV1-12. In one aspect, the self-complementary genome is packaged in an AAV8 capsid. In another aspect, the self-complementary genome is packaged in an AAV2 capsid.


The present invention also relates to a viral vector capable of directing expression of a heterologous gene to RPE and Müller cells of the retina. It is contemplated that the viral vector capsid is an AAV2 or an AAV8 serotype capsid and that the viral vector comprises a vector genome, wherein the heterologous gene is operably linked to an RLBP1 promoter. It is further contemplated that the RLBP1 promoter is the RLBP1 (short) promoter (SEQ ID NO: 3) or the RLBP1 (long) promoter (SEQ ID NO: 10). In another aspect of the invention it is contemplated that the heterologous gene to be expressed in RPE and Müller cells is an RLBP1 coding sequence having for example, the sequence of SEQ ID NO: 6.


The present invention also relates to a viral vector capable of directing expression of a heterologous gene to RPE and Müller cells of the retina, wherein the viral vector capsid is an AAV8 serotype capsid and that the viral vector comprises a self-complementary vector genome wherein a heterologous gene is operably linked to an RLBP1 promoter. It is further contemplated that the RLBP1 promoter is the RLBP1 (short) promoter (SEQ ID NO: 3). In another aspect of the invention it is contemplated that the heterologous gene to be expressed in RPE and Müller cells is an RLBP1 coding sequence having for example, the sequence of SEQ ID NO: 6.


The invention also relates to a composition comprising a viral vector described herein, as well as viral vector compositions in combination with a pharmaceutically acceptable carrier. Specifically, the invention further relates to compositions comprising the viral vectors as described in Table 4. The invention still further relates to compositions comprising viral vectors that can be generated using the plasmids described in Table 2, in conjunction with rAAV production methods known in the art and described herein. The compositions described herein are useful for treating a subject having RLBP1 associated retinal dystrophy and/or improving the rate of dark adaption in a subject having RLBP1-associated retinal dystrophy.


The present invention also relates to nucleic acids that can be used, with the rAAV production methods known in the art and described herein, for the generation of the viral vectors described herein. The invention relates to nucleic acids comprising a gene cassette, wherein the gene cassette comprises, in the 5′ to 3′ direction: (i) a 5′ ITR or a non-resolvable ITR, (ii) a recombinant nucleotide sequence comprising an RLBP1 coding sequence, and (iii) a 3′ ITR. It is contemplated that the nucleic acid may comprise a gene cassette comprising a nucleic acid sequence selected from SEQ ID NOs: 51, 52, 53, 54, and 55. It is contemplated that the nucleic acids of the invention may be plasmids. It is further contemplated that the nucleic acid may be a plasmid comprising a nucleic acid sequence selected from SEQ ID NOs: 26, 27, 28, 29, 30 and 50.


In certain specific aspects of the invention, the nucleic acid can comprise a gene cassette comprising sequences in the 5′ to 3′ direction that are selected from: a) a) SEQ ID NO: 2, 10, 5, 6, 8, and 9, b) SEQ ID NO: 2, 11, 5, 6, 8, 14 and 9, c) SEQ ID NO: 2, 22, 5, 6, 8, 23 and 9, d) SEQ ID NO: 2, 3, 4, 5, 6, 8, 23 and 9, or e) SEQ ID NO: 1, 3, 4, 5, 6, 8, and 9.


The invention also relates to nucleic acids comprising a gene cassette, wherein the gene cassette comprises, in the 5′ to 3′ direction: (i) a 5′ ITR, (ii) a recombinant nucleotide sequence comprising a promoter operably linked to reporter gene, and (iii) a 3′ ITR. It is contemplated that the nucleic acid may comprise a gene cassette comprising a nucleic acid sequence selected from SEQ ID NOs: 56, 57, 59 and 60. It is further contemplated that nucleic acid may be a plasmid comprising a nucleic acid sequence selected from SEQ ID NOs: 31, 32, 34 and 35.


The invention also relates to methods of treating a subject having RLBP1-associated retinal dystrophy wherein the method comprises administering to a subject in need thereof, a composition comprising a viral vector as described herein.


The invention also relates to a method of improving the rate of dark adaption in a subject having RLBP1-associated retinal dystrophy, wherein the method comprises administering to a subject in need thereof, a composition comprising a viral vector as described herein.


The invention still further relates to a method of directing expression of an RLBP1 coding sequence in RPE and Müller cells in the retina of a subject having RLBP1-associated retinal dystrophy, wherein the method comprises the step of contacting the retina of the subject, with a viral vector comprising an AAV8 or AAV2 serotype capsid and a vector genome comprising an RLBP1 coding sequence operably linked to an RLBP1 promoter, such as, for example, the RLBP1(short) (SEQ ID NO: 3) or RLBP1(long) (SEQ ID NO: 10) promoters as described herein.


The invention still further relates to a method of delivering an RLBP1 coding sequence in RPE and Müller cells in the retina of a subject having RLBP1-associated retinal dystrophy, wherein the method comprises the step of contacting the retina of the subject, with a viral vector comprising an AAV8 or AAV2 serotype capsid and a vector genome comprising an RLBP1 coding sequence operably linked to an RLBP1 promoter, such as, for example, the RLBP1(short) (SEQ ID NO: 3) or RLBP1(long) (SEQ ID NO: 10) promoters as described herein.


The invention also includes a viral vector as described in Table 1, or 4, as well as a plasmid described in Table 2.


DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.


The term “capsid” refers to the protein coat of the virus or viral vector. The term “AAV capsid” refers to the protein coat of the adeno-associated virus (AAV), which is composed of a total of 60 subunits; each subunit is an amino acid sequence, which can be viral protein 1(VP1), VP2 or VP3 (Muzyczka N and Berns K I 2001).


The term “gene cassette” refers to a manipulatable fragment of DNA carrying, and capable of expressing, one or more genes, or coding sequences, of interest between one or more sets of restriction sites. A gene cassette can be transferred from one DNA sequence (often in a plasmid vector) to another by ‘cutting’ the fragment out using restriction enzymes and ligating it back into a new context, for example into a new plasmid backbone.


The term “heterologous gene” or “heterologous nucleotide sequence” will typically refer to a gene or nucleotide sequence that is not naturally-occurring in the virus. Alternatively, a heterologous gene or nucleotide sequence may refer to a viral sequence that is placed into a non-naturally occurring environment (e.g.: by association with a promoter with which it is not naturally associated in the virus).


The terms “ITR” or “inverted terminal repeat” refer to the stretch of nucleic acid sequences that exist in Adeno-Associated Viruses (AAV) and/or recombinant Adeno-Associated Viral Vectors (rAAV) that can form a T-shaped palindromic structure, that is required for completing AAV lytic and latent life cycles (Muzyczka N and Berns K I 2001). The term “non-resolvable ITR” refers to a modified ITR such that the resolution by the Rep protein is reduced. A non-resolvable ITR can be an ITR sequence without the terminal resolution site (TRS) which leads to low or no resolution of the non-resolvable ITR and would yield 90-95% of self-complementary AAV vectors (McCarty et al 2003). A specific example of a non-resolvable ITR is “ΔITR”, having a sequence of SEQ ID NO: 1.


The term “operably linked” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, the term refers to the functional relationship of a transcriptional regulatory sequence to a sequence to be transcribed. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribable sequence are contiguous to the transcribable sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.


The term “promoter” refers to a sequence that regulates transcription of an operably-linked gene, or nucleotide sequence encoding a protein, etc. Promoters provide the sequence sufficient to direct transcription, as well as, the recognition sites for RNA polymerase and other transcription factors required for efficient transcription and can direct cell specific expression. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g.: enhancers, kozak sequences and introns). Examples of promoters known in the art and useful in the viral vectors described herein, include the CMV promoter, CBA promoter, smCBA promoter and those promoters derived from an immunoglobulin gene, SV40, or other tissue specific genes (e.g: RLBP1, RPE, VMD2). Specific promoters may also include those described in Table 1, for example, the “RLBP1 (short)” promoter (SEQ ID NO: 3), the “RLBP1 (long)” promoter (SEQ ID NO: 10), RPE65 promoter (SEQ ID NO: 11), VMD2 promoter (SEQ ID NO: 12), and the CMV enhancer+CBA promoter (SEQ ID NO: 22). In addition, standard techniques are known in the art for creating functional promoters by mixing and matching known regulatory elements. “Truncated promoters” may also be generated from promoter fragments or by mix and matching fragments of known regulatory elements; for example the smCBA promoter is a truncated form of the CBA promoter.


The term “RLBP1” refers to the “Retinaldehyde Binding Protein 1”. The human RLBP1 gene is found on chromosome 15 and has the nucleic acid coding sequence as set out in Table 1: SEQ ID NO: 6. The “RLBP1 gene product” is also known as, “cellular retinaldehyde binding protein” or “CRALBP” and is the protein encoded by the RLBP1 gene. The human RLBP1 gene product (hCRALBP) has the amino acid sequence as set out in Table 1: SEQ ID NO: 7. Examples of RLBP1 coding sequences and RLBP1 gene products from other species can be found in Table 1 (e.g.: SEQ ID NOs: 37-48). The term “RLBP1 coding sequence” or “RLBP1 GENE CDS” or “RLBP1 CDS” refers to the nucleic acid sequence that encodes the RLBP1 gene product. One of skill in the art would understand that an RLBP1 coding sequence may include any nucleic acid sequence that encodes an RLBP1 gene product. The RLBP1 coding sequence may or may not include intervening regulatory elements (e.g.: introns, enhancers, or other non-coding sequences).


The term “subject” includes human and non-human animals. Non-human animals include all vertebrates (e.g.: mammals and non-mammals) such as, non-human primates (e.g.: cynomolgus monkey), mice, rats, sheep, dogs, cows, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.


As used herein, the term “treating” or “treatment” of any disease or disorder (e.g., retinitis pigmentosa, RBLP1-associated retinal dystrophy) refers, to ameliorating the disease or disorder such as by slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof. “Treating” or “treatment” can also refer to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. “Treating” or “treatment” can also refer to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. More specifically, “treatment” of RLBP1-associated retinal dystrophy means any action that results in the improvement or preservation of visual function and/or regional anatomy in a subject having RLBP1-associated retinal dystrophy. “Preventing or “prevention” as used herein, refers to preventing or delaying the onset or development or progression of the disease or disorder. “Prevention” as it relates to RLBP1-associated retinal dystrophy means any action that prevents or slows a worsening in visual function, retinal anatomy, and/or an RLBP1-associated retinal dystrophy disease parameter, as described below, in a patient with RLBP1-associated retinal dystrophy and at risk for said worsening. Methods for assessing treatment and/or prevention of disease are known in the art and described herein below.


The term “virus vector” or “viral vector” is intended to refer to a non-wild-type recombinant viral particle (e.g.: a parvovirus, etc.) that functions as a gene delivery vehicle and which comprises a recombinant viral genome packaged within a viral (e.g.: AAV) capsid. A specific type of virus vector may be a “recombinant adeno-associated virus vector”, or “rAAV vector”. The recombinant viral genome packaged in the a viral vector is also referred to herein as the “vector genome”.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1. Relative expression of vector-mediated human RLBP1 mRNA compared to endogenous mouse RLBP1 mRNA in eyes injected with various viral vectors at the dosage of 1×109 (1A) and 1×108 (1B) vector genome (vg) particles per eye.



FIG. 2. Dark adaptation in RLBP1 KO (−/−) and wild-type (+/+) mice.



FIG. 3. Measurement of rate of dark adaptation of RLBP1 KO mice treated with various viral vectors: NVS1 (3B), NVS3 (3A and 3C), NVS3 (3B and 3D), NVS4 (3A and 3C) and NVS5 (3B and 3D).



FIG. 4. Measurement of increased rate of dark adaptation of RLBP1 KO mice treated with various doses of NVS2 and NVS11 is shown in panel 4A. Panel 4B illustrates treatment efficacy of NVS2. Horizontal axis doses are indicated in scientific notation (for example, 3E6=3×106).



FIG. 5. Measurement of increased rate of dark adaptation of RLBP1 KO mice treated with various doses of NVS4 and NVS11 is shown in panel 5A. Panel 5B illustrates treatment efficacy of NVS4. Horizontal axis doses are indicated in scientific notation (for example, 3E6=3×106).



FIG. 6. Measurement of increased rate of dark adaptation of RLBP1 KO mice treated with NVS2 prepared with different purification methods.





DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of viral vectors that express a heterologous gene in RPE and Müller cells of the retina. The invention also relates both to single stranded and self-complementary viral vectors with a heterologous gene expressing the RLBP1 gene product (CRALBP).


Accordingly, the present invention provides recombinant viral vectors that direct expression of the RLBP1 coding sequence to the retina, viral vector compositions, plasmids useful for generating the viral vectors, methods of delivering an RLBP1 coding sequence to the retina, methods of expressing an RLBP1 coding sequence in RPE and Müller cells of the retina, and methods of use of such viral vectors.


Except as otherwise indicated, standard methods known to those skilled in the art may be used for the construction of recombinant parvovirus and rAAV vectors, using recombinant plasmids carrying a viral gene cassette, packaging plasmids expressing the parvovirus rep and/or cap sequences, as well as transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. (e.g.: SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Choi V W et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (2007)).


1. Viral Vectors

The present invention is related to viral vectors that direct expression of a heterologous gene to the retina. In certain aspects of the invention, expression is directed to RPE and Müller cells of the retina. A variety of viral vectors known in the art may be adapted by one of skill in the art for use in the present invention, for example, recombinant adeno-associated viruses, recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, recombinant baculoviruses, etc.


In particular, it is contemplated that the viral vector of the invention may be a recombinant adeno-associated (rAAV) vector. AAVs are small, single-stranded DNA viruses which require helper virus to facilitate efficient replication (Muzyczka N and Berns K I 2001). The viral vector comprises a vector genome and a protein capsid. The viral vector capsid may be supplied from any of the AAV serotypes known in the art, including presently identified human and non-human AAV serotypes and AAV serotypes yet to be identified (See: Choi V W et al 2005, Schmidt et al 2008). Virus capsids may be mixed and matched with other vector components to form a hybrid viral vector, for example the ITRs and capsid of the viral vector may come from different AAV serotypes. In one aspect, the ITRs can be from an AAV2 serotype while the capsid is from, for example, an AAV2 or AAV8 serotype. In addition, one of skill in the art would recognize that the vector capsid may also be a mosaic capsid (e.g.: a capsid composed of a mixture of capsid proteins from different serotypes), or even a chimeric capsid (e.g.: a capsid protein containing a foreign or unrelated protein sequence for generating markers and/or altering tissue tropism). It is contemplated that the viral vector of the invention may comprise an AAV2 capsid. It is further contemplated that the invention may comprise an AAV8 capsid.


The invention relates, in part, to viral vectors wherein the vector genome is single stranded. In certain aspects, the invention is related to a single stranded vector genome comprising, in the 5′ to 3′ direction: (i) a 5′ ITR, (ii) a recombinant nucleotide sequence comprising an RLBP1 coding sequence, and (iii) a 3′ ITR. In certain aspects of the invention the recombinant nucleotide sequence comprises in the 5′ to 3′ direction: (i) a promoter, (ii) an RLBP1 coding sequence, and (iii) an SV40 polyA sequence. In certain aspects, the promoter may be an RLBP1 (short) promoter, an RLBP1 (long) promoter, or a truncated promoter of RLBP1. In particular, the invention relates to a single stranded vector genome comprising a recombinant nucleotide sequence comprising in the 5′ to 3′ direction: an RLBP1 (long) promoter (SEQ ID NO:10), an RLBP1 coding sequence, and an SV40 polyA sequence. In addition, the invention also relates to a single stranded vector genome comprising a recombinant nucleotide sequence comprising in the 5′ to 3′ direction: an RLBP1 (short) promoter (SEQ ID NO: 3), an RLBP1 coding sequence, and an SV40 polyA sequence. Certain aspects of the invention further relate to a single stranded vector genome comprising a recombinant nucleotide sequence packaged in an AAV2 or AAV8 capsid.


In certain aspects of the invention the viral vector comprises an AAV2 capsid (encoded by SEQ ID NO: 18) and a vector genome comprising in the 5′ to 3 direction nucleotide sequences selected from the following: a) SEQ ID NO: 2, 10, 5, 6, 8, and 9; b) SEQ ID NO: 2, 11, 5, 6, 8, 14, 9; c) SEQ ID NO: 2, 22, 5, 6, 8, 23, and 9; and d) SEQ ID NO: 2, 3, 4, 5, 6, 8, 23, and 9. In certain aspects the AAV2 capsid comprises capsid proteins VP1, VP2 and VP3 having an amino acid sequence of SEQ ID NO: 19, 68, and 69, respectively. In certain other aspects the AAV2 capsid may comprise subcombinations of capsid proteins VP1, VP2 and/or VP3.


In certain aspects of the invention the viral vector comprises an AAV8 capsid (encoded by SEQ ID NO: 20) and a vector genome comprising in the 5′ to 3′ direction nucleotide sequences selected from the following: a) SEQ ID NO: 2, 10, 5, 6, 8, and 9; b) SEQ ID NO: 2, 11, 5, 6, 8, 14, 9; c) SEQ ID NO: 2, 22, 5, 6, 8, 23, and 9; and d) SEQ ID NO: 2, 3, 4, 5, 6, 8, 23, and 9. In certain aspects the AAV8 capsid comprises capsid proteins VP1, VP2 and VP3 having an amino acid sequence of SEQ ID NO: 21, 70, and 71. In certain other aspects the AAV8 capsid may comprise subcombinations of capsid proteins VP1, VP2 and/or VP3.


The viral vector can also be an AAV vector comprising a self-complementary genome. Self-complementary rAAV vectors have been previously described in the art (U.S. Pat. No. 7,465,583 and McCarty 2008) and may be adapted for use in the present invention. A self-complementary genome comprises a 5′ ITR and a 3′ ITR (i.e.: resolvable ITR or wild-type ITR) at either end of the genome and a non-resolvable ITR (e.g.: ΔITR, as described herein) interposed between the 5′ and 3′ ITRs. Each portion of the genome (i.e. between each resolvable ITR and non-resolvable ITR) comprises a recombinant nucleotide sequence, wherein each half (i.e.: the first recombinant nucleotide sequence and the second recombinant nucleotide sequence) is complementary to the other, or self-complementary. In other words, the self-complementary vector genome is essentially an inverted repeat with the two halves joined by the non-resolvable ITR. In certain aspects the invention is related to a self-complementary vector genome comprising, in the 5′ to 3′ direction, (i) a 5′ ITR, (ii) a first recombinant nucleotide sequence, (iii) a non-resolvable ITR, (iv) a second recombinant nucleotide sequence, and (v) a 3′ ITR. In a certain aspect of the invention the second recombinant nucleotide sequence of the vector genome comprises, an RLBP1 promoter, an RLBP1 coding sequence, and an SV40 polyA sequence and the first recombinant nucleotide sequence is self-complementary to the second nucleotide sequence. In certain specific aspects the RLBP1 promoter has the nucleotide sequence of SEQ ID NO: 3. In certain aspects of the invention, the second recombinant nucleotide sequence comprises nucleic acid sequences in the 5′ to 3′ direction of SEQ ID NO: 3, 4, 5, 6, and 8 and the first recombinant nucleotide sequence comprises sequences that are self-complementary to, or the reverse complement of, the second recombinant sequence, for example, SEQ ID NOs: 62, 63, 64, 65, and 66. It is also contemplated that the viral vector of the invention may comprise a self-complementary genome wherein the first recombinant nucleotide sequence of the vector genome comprises, an RLBP1 promoter, an RLBP1 coding sequence, and an SV40 polyA sequence and the second recombinant nucleotide sequence is self-complementary to the first recombinant nucleotide sequence.


In certain aspects of the invention the self-complementary viral vector comprises an AAV2 capsid (encoded by SEQ ID NO: 18) and a vector genome comprising a nucleotide sequence comprising sequences in the 5′ to 3′ direction SEQ ID NO: 36, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. In certain aspects the AAV2 capsid comprises capsid proteins VP1, VP2 and VP3 having an amino acid sequence of SEQ ID NO: 19, 68, and 69, respectively. In certain other aspects the AAV2 capsid may comprise subcombinations of capsid proteins VP1, VP2 and/or VP3.


In certain aspects of the invention the self-complementary viral vector comprises an AAV8 capsid (encoded by SEQ ID NO: 20) and a vector genome comprising a nucleotide sequence comprising sequences in the 5′ to 3′ direction SEQ ID NO: 36, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. In certain aspects the AAV8 capsid comprises capsid proteins VP1, VP2 and VP3 having an amino acid sequence of SEQ ID NO: 21, 70, and 71. In certain other aspects the AAV8 capsid may comprise subcombinations of capsid proteins VP1, VP2 and/or VP3.


Thus, the invention also relates to viral vectors as described herein, comprising a truncated promoter of RLBP1.


The invention further relates to a viral vector that directs expression of a heterologous gene to RPE and Müller cells of the retina, wherein the viral vector comprises an AAV8 capsid and a vector genome comprising an RLBP1 (short) promoter (SEQ ID NO:3) operably linked to a heterologous gene. In certain aspects of the invention, the vector genome is a self-complementary genome.


The invention also relates to methods of expressing RLBP1 in RPE cells and Müller cells of the retina. In certain aspects of the invention the method comprises contacting the retinal cells with a viral vector comprising an AAV capsid and a vector genome comprising an RLBP1 coding sequence operably linked to an RLBP1 promoter, which may be an RLBP1 (short) promoter (SEQ ID NO:3). In certain aspects of the invention the AAV capsid is AAV2. In certain other aspects, the AAV capsid is AAV8. In other aspects of the invention the method comprises contacting the retinal cells with a viral vector comprising an AAV capsid and a vector genome comprising an RLBP1 coding sequence operably linked to an RLBP1 promoter, which may be an RLBP1 (long) promoter (SEQ ID NO: 10). In certain aspects of the invention the AAV capsid is AAV2. In certain other aspects, the AAV capsid is AAV8.


Methods for generating viral vectors are well known in the art and would allow for the skilled artisan to generate the viral vectors of the invention (see, e.g., U.S. Pat. No. 7,465,583), including the viral vectors described in Table 4, using the plasmids described in Table 2 and the Examples.


In general, methods of producing rAAV vectors are applicable to producing the viral vectors of the invention; the primary difference between the methods is the structure of the genetic elements to be packaged. To produce a viral vector according to the present invention, sequences of the genetic elements and plasmids as described in table 2 can be used to produce the encapsidated viral genome.


The genetic elements as described in table 2 are in the context of a circular plasmid, but one of skill in the art will appreciated that a DNA substrate may be provided in any form known in the art, including but not limited to a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or a viral vector (e.g., adenovirus, herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral vectors, and the like). Alternatively, the genetic elements in table 2 necessary to produce the viral vectors described herein may be stably incorporated into the genome of a packaging cell.


The viral vector particles according to the invention may be produced by any method known in the art, e.g., by introducing the sequences to be replicated and packaged into a permissive or packaging cell, as those terms are understood in the art (e.g., a “permissive” cell can be infected or transduced by the virus; a “packaging” cell is a stably transformed cell providing helper functions).


In one embodiment, a method is provided for producing an RLBP1 viral vector, wherein the method comprises providing to a cell permissive for parvovirus replication: (a) a nucleotide sequence containing the genetic elements for producing a vector genome of the invention (as described in detail below and in table 2); (b) nucleotide sequences sufficient for replication of the vector genome sequence in (a) to produce a vector genome; (c) nucleotide sequences sufficient to package the vector genome into a parvovirus capsid, under conditions sufficient for virus vectors comprising the vector genome encapsidated within the parvovirus capsid to be produced in the cell. Preferably, the parvovirus replication and/or capsid coding sequences are AAV sequences.


Any method of introducing the nucleotide sequence carrying the gene cassettes described below into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.


Viral vectors described herein may be produced using methods known in the art, such as, for example, triple transfection or baculovirus mediated virus production. Any suitable permissive or packaging cell known in the art may be employed to produce the vectors. Mammalian cells are preferred. Also preferred are trans-complementing packaging cell lines that provide functions deleted from a replication-defective helper virus, e.g., 293 cells or other E1a trans-complementing cells. Also preferred are mammalian cells or cell lines that are defective for DNA repair as known in the art, as these cell lines will be impaired in their ability to correct the mutations introduced into the plasmids described herein.


The gene cassette may contain some or all of the parvovirus (e.g., AAV) cap and rep genes. Preferably, however, some or all of the cap and rep functions are provided in trans by introducing a packaging vector(s) encoding the capsid and/or Rep proteins into the cell. Most preferably, the gene cassette does not encode the capsid or Rep proteins. Alternatively, a packaging cell line is used that is stably transformed to express the cap and/or rep genes (see, e.g., Gao et al., (1998) Human Gene Therapy 9:2353; Inoue et al., (1998) J. Virol. 72:7024; U.S. Pat. No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785; WO 96/17947).


In addition, helper virus functions are preferably provided for the virus vector to propagate new virus particles. Both adenovirus and herpes simplex virus may serve as helper viruses for AAV. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers). Exemplary helper viruses include, but are not limited to, Herpes simplex (HSV) varicella zoster, cytomegalovirus, and Epstein-Barr virus. The multiplicity of infection (MOI) and the duration of the infection will depend on the type of virus used and the packaging cell line employed. Any suitable helper vector may be employed. Preferably, the helper vector is a plasmid, for example, as described by Xiao et al., (1998) J. Virology 72:2224. The vector can be introduced into the packaging cell by any suitable method known in the art, as described above.


Vector stocks free of contaminating helper virus may be obtained by any method known in the art. For example, recombinant single stranded or self complementary virus and helper virus may be readily differentiated based on size. The viruses may also be separated away from helper virus based on affinity for a heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973). Preferably, deleted replication-defective helper viruses are used so that any contaminating helper virus is not replication competent. As a further alternative, an adenovirus helper lacking late gene expression may be employed, as only adenovirus early gene expression is required to mediate packaging of the duplexed virus. Adenovirus mutants defective for late gene expression are known in the art (e.g., ts100K and ts149 adenovirus mutants).


One method for providing helper functions employs a non-infectious adenovirus miniplasmid that carries all of the helper genes required for efficient AAV production (Ferrari et al., (1997) Nature Med. 3:1295; Xiao et al., (1998) J. Virology 72:2224). The rAAV titers obtained with adenovirus miniplasmids are forty-fold higher than those obtained with conventional methods of wild-type adenovirus infection (Xiao et al., (1998) J. Virology 72:2224). This approach obviates the need to perform co-transfections with adenovirus (Holscher et al., (1994), J. Virology 68:7169; Clark et al., (1995) Hum. Gene Ther. 6:1329; Trempe and Yang, (1993), in, Fifth Parvovirus Workshop, Crystal River, Fla.).


Other methods of producing rAAV stocks have been described, including but not limited to, methods that split the rep and cap genes onto separate expression cassettes to prevent the generation of replication-competent AAV (see, e.g., Allen et al., (1997) J. Virol. 71:6816), methods employing packaging cell lines (see, e.g., Gao et al., (1998) Human Gene Therapy 9:2353; Inoue et al., (1998) J. Virol. 72:7024; U.S. Pat. No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785; WO 96/17947), and other helper virus free systems (see, e.g., U.S. Pat. No. 5,945,335 to Colosi).


Herpesvirus may also be used as a helper virus in AAV packaging methods. Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageously facilitate for more scalable AAV vector production schemes. A hybrid herpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep and cap genes has been described (Conway et al., (1999) Gene Therapy 6:986 and WO 00/17377).


In summary, the gene cassette to be replicated and packaged, parvovirus cap genes, appropriate parvovirus rep genes, and (preferably) helper functions are provided to a cell (e.g., a permissive or packaging cell) to produce rAAV particles carrying the vector genome. The combined expression of the rep and cap genes encoded by the gene cassette and/or the packaging vector(s) and/or the stably transformed packaging cell results in the production of a viral vector particle in which a viral vector capsid packages a viral vector genome according to the invention. The single stranded or self-complementary viral vectors are allowed to assemble within the cell, and may then be recovered by any method known by those of skill in the art and described in the examples. For example, viral vectors may be purified by standard CsCl centrifugation methods (Grieger J C et al 2006) or by various methods of column chromatography known to the skilled artisan (see: Lock M et al (2010), Smith R H et al (2009) and Vadenberghe L H et al (2010)).


The reagents and methods disclosed herein may be employed to produce high-titer stocks of the inventive viral vectors, preferably at essentially wild-type titers. It is also preferred that the parvovirus stock has a titer of at least about 105 transducing units (tu)/ml, more preferably at least about 106 to/ml, more preferably at least about 107 to/ml, yet more preferably at least about 108 to/ml, yet more preferably at least about 109 to/ml, still yet more preferably at least about 1010 to/ml, still more preferably at least about 10″ to/ml, or more.


Further, the RLBP1 viral vectors of the invention, may have an improved transducing unit/particle ratio over conventional AAV vectors. Preferably, the tu/particle ratio is less than about 1:50, less than about 1:20, less than about 1:15, less than about 1:10, less than about 1:8, less than about 1:7, less than about 1:6, less than about 1:5, less than about 1:4, or lower. Typically, the tu/particle ratio will be greater than about 1:1, 1:2, 1:3 or 1:4.


2. Nucleic Acids for Use in Generating the Viral Vector

The invention also relates to nucleic acids useful for the generation of viral vectors. In certain aspects of the invention, the nucleic acids useful for the generation of viral vectors may be in the form of plasmids. Plasmids useful for the generation of viral vectors, also referred to as a viral vector plasmid, may contain a gene cassette. At a minimum, a gene cassette of a viral vector plasmid contains: a heterologous gene and its regulatory elements (e.g.: promoter, enhancer, and/or introns, etc.), and 5′ and 3′ AAV inverted terminal repeats (ITRs).


The composition of the heterologous gene and its regulatory elements will depend upon the use to which the resulting vector will be put. For example, one type of heterologous gene sequence includes a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc. For example, where the reporter sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for beta-galactosidase activity. Where the reporter sequence is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.


The heterologous gene sequences, when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.


The heterologous gene may also be a non-marker sequence encoding a product which is useful in biology and medicine, such as proteins, peptides, RNA, enzymes, dominant negative mutants, or catalytic RNAs. Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNAs. One example of a useful RNA sequence is a sequence which inhibits or extinguishes expression of a targeted nucleic acid sequence in the treated animal.


The heterologous gene may also be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed. It is contemplated in the present invention that the heterologous gene sequence may be an RLBP1 coding sequence. Examples of RLBP1 coding sequences are provided in Table 1: SEQ ID NOs: 6, 37, 39, 41, 43, 45 or 47.


In addition to the heterologous gene, the gene cassette may include regulatory elements operably linked to the heterologous gene. These regulatory elements may include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency; sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of regulatory sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized. Regulatory element sequences of the invention include those described in Table 1, for example SEQ ID NO: 3, 4, 5, 8, 10, 11, 12 and 22.


The gene cassette may include an RLBP1 promoter with a nucleic acid sequence of SEQ ID NO: 3 or 10 operably linked to a heterologous gene. In particular, the RLBP1 short promoter (SEQ ID NO: 3) is operably linked to an RLBP1 coding sequence (SEQ ID NO: 6, 37, 39, 41, 43, 45 or 47). Alternatively, the RLBP1 long promoter (SEQ ID NO: 10) is operably linked to an RLBP1 coding sequence (SEQ ID NO: 6, 37, 39, 41, 43, 45 or 47).


It is contemplated that the ITRs of AAV serotype 2 may be used (e.g.: SEQ ID NO: 2, 9, 16, 17, 36). However, ITRs from other suitable serotypes may be selected from among any AAV serotype known in the art, as described herein. These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from any AAV serotype known, or yet to be identified serotypes, for example, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like. Alternatively, such AAV components may also be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.).


It is contemplated that in certain aspects of the invention, one ITR of the gene cassette may be a modified ITR, or non-resolvable ITR, sequence without the terminal resolution site (TRS). During replication of a gene cassette comprising a non-resolvable ITR, the inability of Rep protein to resolve the non-resolvable ITRs will result in a dimeric inverted repeat sequence (i.e.: self-complementary) with a non-resolvable ITR (e.g.: ΔITR) in the middle and a wild-type ITR at each end. The resulting sequence is a self-complementary viral genome sequence such that the genome is capable of forming a hairpin structure upon release from the capsid (see also: U.S. Pat. No. 7,465,583 and McCarty (2008)) A non-resolvable ITR may be produced by any method known in the art. For example, insertion into the ITR will displace the TRS and result in a non-resolvable ITR. Preferably, the insertion is in the region of the TRS site. Alternatively, the ITR may be rendered non-resolvable by deletion of the TRS site, a specific example includes ΔITR (SEQ ID NO: 1).


The invention relates to nucleic acids that comprise a gene cassette comprising in the 5′ to 3′ direction nucleic acid sequences selected from the following: a) SEQ ID NOs: 2, 10, 5, 6, 8, and 9; b) SEQ ID NOs: 2, 11, 5, 6, 8, 14 and 9; c) SEQ ID NOs: 2, 22, 5, 6, 8, 23 and 9; d) SEQ ID NOs: 2, 3, 4, 5, 6, 8, 23 and 9; e) SEQ ID NOs: 2, 10, 5, 24, 8, and 9; f) SEQ ID NOs: 2, 11, 24, 8, 14, and 9; and g) SEQ ID NOs: 2, 12, 24, 8, 14, and 9. In certain aspects the nucleic acid comprising the gene cassette may be a plasmid. In particular, the sequence of the plasmid may have a sequence selected from SEQ ID NOs: 27, 28, 29, 30, 32, 33, 34 and 35.


The invention also relates to nucleic acids that comprise a gene cassette comprising in the 5′ to 3′ direction nucleic acid sequences selected from the following: a) SEQ ID NOs: 1, 3, 4, 5, 6, 8, and 9; and b) SEQ ID NOs: 1, 3, 4, 5, 24, 8 and 9. In certain aspects the nucleic acid comprising the gene cassette may be a plasmid. In particular, the sequence of the plasmid may have a sequence selected from SEQ ID NOs: 26, 31 and 50.


Methods for incorporating the elements in Table 2 are well known in the art and would allow for the skilled artisan to generate the nucleic acids and plasmids of the invention using the methods outlined in Table 3 and the Examples.


3 Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising the viral vectors of the invention formulated together with a pharmaceutically acceptable carrier. The compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing, for example, RLBP1-associated retinal dystrophy, and/or retinal pigmentosa (RP). Pharmaceutically acceptable carriers enhance or stabilize the composition, or can be used to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, surfactants, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.


A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. It is preferred that administration be subretinal. The pharmaceutically acceptable carrier should be suitable for subretinal, intravitreal, intravenous, sub-cutaneous or topical administration.


The composition should be sterile and fluid. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.


Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the viral vector is employed in the pharmaceutical compositions of the invention. The viral vectors may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.


Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.


A physician or veterinarian can start doses of the viral vectors of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions of the present invention, for the treatment of RLBP1-associated retinal dystrophy as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. For subretinal administration with a viral vector, the dosage may range from 1×108 vector genomes (vg)/eye to 1×1012 vg/eye. For example the dosage may be, 1×108 vg/eye, 2.5×108 vg/eye, 5×108 vg/eye, 7.5×108 vg/eye, 1×109 vg/eye, 2.5×109 vg/eye, 5×109 vg/eye, 7.5×109 vg/eye, 1×1010 vg/eye, 2.5×1010 vg/eye, 5×1010 vg/eye, 7.5×1010 vg/eye, 1×1011 vg/eye, 2.5×1011 vg/eye, 5×1011 vg/eye, 7.5×1011 vg/eye, 1×1012 vg/eye.


The viral vectors described herein are mainly used as one time doses per eye, with the possibility of repeat dosing to treat regions of the retina that are not covered in the previous dosing. The dosage of administration may vary depending on whether the treatment is prophylactic or therapeutic.


The various features and embodiments of the present invention, referred to in individual sections and embodiments above apply, as appropriate, to other sections and embodiments, mutatis mutandis. Consequently features specified in one section or embodiment may be combined with features specified in other sections or embodiments, as appropriate.


4. Therapeutic Uses

Viral vectors as described herein, can be used at a therapeutically useful concentration for the treatment of eye related diseases, by administering to a subject in need thereof, an effective amount of the viral vectors of the invention. More specifically, the present invention provides a method of treating RLBP1-associated retinal dystrophy, by administering to a subject in need thereof an effective amount of a viral vector comprising an RLBP1 coding sequence.


The present invention provides a viral vector comprising an RLBP1 coding sequence for use in treating RLBP1-associated retinal dystrophy in a subject.


Table A: RLBP1 Mutations and Associated Phenotypes of RLBP1-Associated Retinal Dystrophy.


Disease phenotypes of RLBP1-associated retinal dystrophy include: Autosomal recessive retinitis pigmentosa (AARP), Bothnia dystrophy (BD), Newfoundland rod-cone dystrophy (NFRCD), Retinitis punctata albescens (RPA) and Fundus albipunctatus (FA).

















TABLE A











Pigment




# pts
Mutation
Region
Disease
Night Blind
Yellow Dots
Deposits
Atrophy
Reference















Missense Mutations















67 
R234W
Sweden
BD
Yes
Perifoveal, midperiphery
In advanced
Advanced
Burstedt et al 2001;










Golovleva et al 2010;










Golovleva et al 2012


10 
R234W/M226K
Sweden
BD
Yes
Perifoveal, midperiphery
In advanced
Advanced
Köhn et al 2008;










Golovleva et al 2010;










Golovleva et al 2012


2
M226K
Sweden
BD
Yes
Perifoveal, midperiphery
In advanced
Advanced
Golovleva et al 2010;










Golovleva et al 2012


4
G116R
Pakistan
FA
Yes
Midperiphery
No
No
Naz et al 2011


4
R151Q
Saudi Arabia
FA
Yes
Whole fundus
No
No
Katsaris et al 2001


4
R151Q
India
ARRP
Yes
Whole fundus
Yes
Yes
Maw et al 1997


1
R234W
Japan
BD
Yes
Perifoveal, midperiphery
In advanced
Advanced
Nojima et al 2011


1
R103W R234W
Japan
RPA
Yes
Perifoveal, midperiphery
In advanced
Yes
Nakamura et al 2011


1
G146D I201T
USA
RPA
No
Midperiphery
No
No
Demirci et al 2004


1
R103W
USA
RPA
Yes
Midperiphery
No
Yes
Demirci et al 2004







Truncating Mutations















26 
324G_A IVS3_2T 3 C
Canada
NFRCD
Yes
Perifoveal, midperiphery
No
Yes
Eichers et al 2002


6
R156X
Pakistan
FA
Yes
Midperiphery
No
No
Naz et al 2011


4
R151W Gly31 (2-bp del)
USA
RPA
Yes
Midperiphery
Few, peripheral
No
Fishman et al 2004


6
Exons 7_9 del
Morocco
RPA
Yes
Perifoveal, midperiphery
No
No
Humbert et al 2006










Littink et al 2012


1
IVS3_2T 3 C M226K
USA
RPA
Yes
Perifoveal, midperiphery
No
No
Morimura et al 1999


1
Q278(1-bp del)
USA
RPA
Yes
Perifoveal
Few, peripheral
Yes
Morimura et al 1999









Use of recombinant AAV has been shown to be feasible and safe for the treatment of retinal disease (See, e.g., Bainbridge et al. 2008, Houswirth et al 2008, Maguire et al 2008). The viral vectors of the invention can be used, inter alia, to treat and prevent progression of RLBP1-associated retinal dystrophy and improve vision loss. Viral vectors of the invention can also be used in patients where other retinal dystrophy is caused by other loss of function mutations in the RLBP1 gene, for example, Autosomal recessive retinitis pigmentosa, Retinitis punctata albescens and Fundus albipunctatus.


The present invention is also relates to a method of expressing an RLBP1 coding sequence in RPE and Müller cells of the retina, by administering viral vectors of the invention to a subject in need thereof. The present invention also relates to viral vectors of the invention for use in expressing an RLBP1 coding sequence in RPE and/or Müller cells of the retina of the subject in need thereof. The invention also contemplates a method of delivering an RLBP1 coding sequence to the retina, specifically to RPE and/or Müller cells in the retina, of a subject having RLBP1-associated retinal dystrophy. It is contemplated that the an RLBP1 coding sequence is delivered to the subject in need thereof by contacting the retina,


RPE and/or Müller cells of the subject with a viral vector as described herein. Alternatively, an RLBP1 coding sequence is delivered to a subject by administering to the subject a viral vector as described herein.


The present invention further includes methods of expressing an RLBP1 coding sequence in RPE and/or Müller cells in the retina of a subject having RLBP1-associated retinal dystrophy, by contacting the retina of the subject with viral vectors of the invention. In certain aspects RPE and/or Müller cells of the retina of the subject are contacted with viral vectors of the invention.


It is further contemplated that the viral vectors used in the methods described herein comprise an AAV2 or AAV8 capsid, and the vector genome comprises an RLBP1 coding sequence operably linked to an RLBP1 promoter with a nucleotide sequence selected from SEQ ID NO: 3 or 10. It is further contemplated that the vector genome can be self-complementary.


In one aspect the viral vectors described herein can be administered subretinally or intravitreally using methods known to those of skill in the art.


Treatment and/or prevention of ocular disease such as RLBP1-associated retinal dystrophy can be determined by an ophthalmologist or health care professional using clinically relevant measurements of visual function and/or retinal anatomy. Treatment of RLBP1-associated retinal dystrophy means any action (e.g., administration of a viral vector described herein) contemplated to improve or preserve visual function and/or retinal anatomy. In addition, prevention as it relates to RLBP1-associated retinal dystrophy means any action (e.g., administration of a viral vector described herein) that prevents or slows a worsening in visual function, retinal anatomy, and/or RLBP1-associated retinal dystrophy disease phenotype, as defined herein, in a patient at risk for said worsening.


Visual function may include, for example, visual acuity, visual acuity with low illumination, visual field, central visual field, peripheral vision, contrast sensitivity, dark adaptation, photostress recovery, color discrimination, reading speed, dependence on assistive devices (e.g., large typeface, magnifying devices, telescopes), facial recognition, proficiency at operating a motor vehicle, ability to perform one or more activities of daily living, and/or patient-reported satisfaction related to visual function. Thus, treatment of retinitis pigmentosa (RP), specifically RLBP1-associated retinal dystrophy, can be said to occur where a subject has an at least 10% decrease or lack of a 10% or more increase in time to a pre-specified degree of dark adaptation. In addition, treatment of RLBP1-associated retinal dystrophy can be said to occur where a subject exhibits early severe night blindness and slow dark adaptation in young age, followed by progressive loss of visual acuity, visual fields and color vision, leading to legal blindness, determined by a qualified health care professional (i.e., ophthalmologist) (Burstedt and Mönestam, 2010).


Exemplary measures of visual function include Snellen visual acuity, ETDRS visual acuity, low-luminance visual acuity, Amsler grid, Goldmann visual field, standard automated perimetry, microperimetry, Pelli-Robson charts, SKILL card, Ishihara color plates, Farnsworth D15 or D100 color test, standard electroretinography, multifocal electroretinography, validated tests for reading speed, facial recognition, driving simulations, and patient reported satisfaction. Thus, treatment of RLBP1-associated retinal dystrophy can be said to be achieved upon a gain of or failure to lose 2 or more lines (or 10 letters) of vision on an ETDRS scale. In addition, treatment of RLBP1-associated retinal dystrophy can be said to occur where a subject exhibits at least a 10% increase or lack of 10% decrease in reading speed (words per minute). In addition, treatment of RLBP1-associated retinal dystrophy can be said to occur where a subject exhibits at least a 20% increase or lack of a 20% decrease in the proportion of correctly identified plates on an Ishihara test or correctly sequenced disks on a Farnsworth test. Thus, treatment of, for example, RLBP1-associated retinal dystrophy can be determined by, for example, improvement of rate of dark adaptation, or an improvement in, or slowing of the rate of, visual acuity loss.


Undesirable aspects of retinal anatomy that may be treated or prevented include, for example, retinal atrophy, retinal pigment epithelium atrophy, narrowing of retinal vessels, pigmentary clumping, retinal yellow/white spots, subretinal fluid.


Exemplary means of assessing retinal anatomy include fundoscopy, fundus photography, fluorescein angiography, indocyanine green angiography, optical coherence tomography (OCT), spectral domain optical coherence tomography, scanning laser ophthalmoscopy, confocal microscopy, adaptive optics, fundus autofluorescence, biopsy, necropsy, and immunohistochemistry. Thus, RLBP1-associated retinal dystrophy can be said to be treated in a subject as determined by, for example, a reduction in the rate of development of retinal atrophy.


Subjects to be treated with therapeutic agents of the present invention can also be administered other therapeutic agents or devices with known efficacy for treating retinal dystrophy such as vitamin and mineral preparations, low-vision aids, guide dogs, or other devices known to assist patients with low vision.


Currently there are no other approved therapeutic agents for the treatment of RLBP1-associated retinal dystrophy. As other new therapies emerge, the two can be administered sequentially in either order or simultaneously as clinically indicated.


EXAMPLES

The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims.


Example 1
Construction of AAV-ITR Plasmids
1.1 Cloning of AAV-ITR Plasmids:

The nucleic acid sequences of the individual plasmid elements are described in Table 1. The sequences were either synthesized or purchased commercially. Table 2 describes the elements that exist in each plasmid that was constructed. Standard molecular biology cloning techniques were used in generating the plasmids as described in Table 3. The plasmid backbone pAAV-MCS (Stratagene®) with Ampicillin resistance or pUC57 with Kanamycin resistance was used as the backbone and starting material. The individual sequence elements were cloned in at restriction enzyme sites or using blunt end cloning.


Because the antibiotic resistance gene cassette contained in the plasmid backbone does not play a role in the production of the AAV vectors, one of skill in the art could use alternate plasmid backbones and/or antibiotic resistance gene cassettes and yield the same viral vectors. We have demonstrated that functionally equivalent NVS2 vectors can be generated using plasmids with different backbones. For example, plasmid sequences SEQ ID NO: 26 and SEQ ID NO: 50 produce functionally equivalent NVS2 vectors.


1.2. Triple Plasmid Transfection to Produce rAAV Vectors:


Recombinant AAV (rAAV) viral vectors were generated by triple transfection methods. Methods for triple transfection are known in the art (Ferrari F K et al 1997). Briefly, AAV-ITR-containing plasmids (described in Table 2), AAV-RepCap containing plasmid (carrying Rep2 and Cap2 or Cap8) and Adeno-helper plasmid (carrying genes that assist in completing AAV replication cycle) were co-transfected into 293 cells. Cells were cultured for 4 days. At the end of the culture period the cells were lysed and the vectors in the culture supernatant and in the cell lysate were purified by a standard CsCl gradient centrifugation method (method modified based on Grieger J C et al 2006). The purified viral vectors are described in Table 4.


Alternatively, GMP-like rAAV vectors were generated by the cell transfection and culture methods described above. The harvested cell culture material was then processed by column chromatography based on methods described by Lock M et al (2010), Smith R H et al (2009) and Vadenberghe L H et al (2010).


1.3. Variation of 5′ ITR Sequences:

As described previously (Samulski et al, 1983; Muzyczka et al, 1984), mutations within the terminal repeat sequences of AAV plasmids are well tolerated in generating functional AAV vectors. Even plasmids with one of the two ITRs deleted, the AAV sequences could be rescued, replicated, and infectious virions be produced, as long as the existing ITR in the construct contains the full AAV ITR sequence (Samulski et al, 1983; Muzyczka et al, 1984). Therefore, even though SEQ. ID. NO. 2 is used as the 5′ ITR sequence of all single-stranded AAV vectors described in this document, it is expected that any 5′ITR sequence that carries the terminal resolution site (i.e.: SEQ. ID. NOS. 2, 16 and 17) would produce vectors with the same functionality.









TABLE 1







Sequence of viral vector and plasmid elements


AMINO ACID SEQUENCE OR POLYNUCLEOTIDE (PN)








SEQUENCE



ELEMENTS
SEQUENCE IDENTIFIER (SEQ. ID. NO:) AND SEQUENCE





ΔITR
 1



cgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcg



tcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgc



gcagagagggagtgg





5′ ITR
 2



ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcga



cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagag



ggagtggccaactccatcactaggggttcct





Human RLBP1
 3


Promoter(short)
ttgtcctctccctgcttggccttaaccagccacatttctcaact


(NT_010274.17)
gaccccactcactgcagaggtgaaaactaccatgccaggtcctg



ctggctgggggaggggtgggcaataggcctggatttgccagagc



tgccactgtagatgtagtcatatttacgatttcccttcacctct



tattaccctggtggtggtggtgggggggggggggtgctctctca



gcaaccccaccccgggatcttgaggagaaagagggcagagaaaa



gagggaatgggactggcccagatcccagccccacagccgggctt



ccacatggccgagcaggaactccagagcaggagcacacaaagga



gggctttgatgcgcctccagccaggcccaggcctctcccctctc



ccctttctctctgggtcttcctttgccccactgagggcctcctg



tgagcccgatttaacggaaactgtgggcggtgagaagttcctta



tgacacactaatcccaacctgctgaccggaccacgcctccagcg



gagggaacctctagagctccaggacattcaggtaccaggtagcc



ccaaggaggagctgccga





MODIFIED
 4


SV40INTRON
aactgaaaaaccagaaagttaactggtaagtttagtctttttgt


(MODIFIED
cttttatttcaggtcccggatccggtggtggtgcaaatcaaaga


EF579804)
actgctcctcagtggatgttgcctttacttctaggcctgtacgg



aagtgttacttctgctctaaaagctgcggaattgtacccgcccc



gggatcc





ADDED-KOZAK
 5



gccacc





HUMAN RLBP1
 6


GENE CDS
atgtcagaaggggtgggcacgttccgcatggtacctgaagagga


NM_000326.4
acaggagctccgtgcccaactggagcagctcacaaccaaggacc



atggacctgtctttggcccgtgcagccagctgccccgccacacc



ttgcagaaggccaaggatgagctgaacgagagagaggagacccg



ggaggaggcagtgcgagagctgcaggagatggtgcaggcgcagg



cggcctcgggggaggagctggcggtggccgtggcggagagggtg



caagagaaggacagcggcttcttcctgcgcttcatccgcgcacg



gaagttcaacgtgggccgtgcctatgagctgctcagaggctatg



tgaatttccggctgcagtaccctgagctctttgacagcctgtcc



ccagaggctgtccgctgcaccattgaagctggctaccctggtgt



cctctctagtcgggacaagtatggccgagtggtcatgctcttca



acattgagaactggcaaagtcaagaaatcacctttgatgagatc



ttgcaggcatattgcttcatcctggagaagctgctggagaatga



ggaaactcaaatcaatggcttctgcatcattgagaacttcaagg



gctttaccatgcagcaggctgctagtctccggacttcagatctc



aggaagatggtggacatgctccaggattccttcccagcccggtt



caaagccatccacttcatccaccagccatggtacttcaccacga



cctacaatgtggtcaagcccttcttgaagagcaagctgcttgag



agggtctttgtccacggggatgacctttctggtttctaccagga



gatcgatgagaacatcctgccctctgacttcgggggcacgctgc



ccaagtatgatggcaaggccgttgctgagcagctctttggcccc



caggcccaagctgagaacacagccttctga





HUMAN RLBP1
 7


GENE PRODUCT
MSEGVGTFRMVPEEEQELRAQLEQLTTKDHGPVFGPCSQLPRHT


(CELLULAR
LQKAKDELNEREETREEAVRELQEMVQAQAASGEELAVAVAERV


RETINALDEHYDE
QEKDSGFFLRFIRARKFNVGRAYELLRGYVNFRLQYPELFDSLS


BINDING PROTEIN -
PEAVRCTIEAGYPGVLSSRDKYGRVVMLFNIENWQSQEITFDEI


CRALBP)
LQAYCFILEKLLENEETQINGFCIIENFKGFTMQQAASLRTSDL



RKMVDMLQDSFPARFKAIHFIHQPWYFTTTYNVVKPFLKSKLLE



RVFVHGDDLSGFYQEIDENILPSDFGGTLPKYDGKAVAEQLFGP



QAQAENTAF





SV40 POLYA
 8


(EF579804)
gatcataatcagccataccacatttgtagaggttttacttgctt



taaaaaacctcccacacctccccctgaacctgaaacataaaatg



aatgcaattgttgttgttaacttgtttattgcagcttataatgg



ttacaaataaagcaatagcatcacaaatttcacaaataaagcat



ttttttcactgcattctagttgtggtttgtccaaactcatcaat



gtatcttatcatgtct





3′ ITR
 9


(AF043303)
aggaacccctagtgatggagttggccactccctctctgcgcgct



cgctcgctcactgaggccgggcgaccaaaggtcgcccgacgccc



gggctttgcccgggcggcctcagtgagcgagcgagcgcgcag





Human RLBP1
10


Promoter(long)
ttgtcctctccctgcttggccttaaccagccacatttctcaact


(NT_010274.17)
gaccccactcactgcagaggtgaaaactaccatgccaggtcctg



ctggctgggggaggggtgggcaataggcctggatttgccagagc



tgccactgtagatgtagtcatatttacgatttcccttcacctct



tattaccctggtggtggtggtgggggggggggggtgctctctca



gcaaccccaccccgggatcttgaggagaaagagggcagagaaaa



gagggaatgggactggcccagatcccagccccacagccgggctt



ccacatggccgagcaggaactccagagcaggagcacacaaagga



gggctttgatgcgcctccagccaggcccaggcctctcccctctc



ccctttctctctgggtcttcctttgccccactgagggcctcctg



tgagcccgatttaacggaaactgtgggcggtgagaagttcctta



tgacacactaatcccaacctgctgaccggaccacgcctccagcg



gagggaacctctagagctccaggacattcaggtaccaggtagcc



ccaaggaggagctgccgacctggcaggtaagtcaatacctgggg



cttgcctgggccagggagcccaggactggggtgaggactcaggg



gagcagggagaccacgtcccaagatgcctgtaaaactgaaacca



cctggccattctccaggttgagccagaccaatttgatggcagat



ttagcaaataaaaatacaggacacccagttaaatgtgaatttca



gatgaacagcaaatacttttttagtattaaaaaagttcacattt



aggctcacgcctgtaatcccagcactttgggaggccgaggcagg



cagatcacctgaggtcaggagttcgagaccagcctggccaacat



ggtgaaaccccatctccactaaaaataccaaaaattagccaggc



gtgctggtgggcacctgtagttccagctactcaggaggctaagg



caggagaattgcttgaacctgggaggcagaggttgcagtgagct



gagatcgcaccattgcactctagcctgggcgacaagaacaaaac



tccatctcaaaaaaaaaaaaaaaaaaaaagttcacatttaactg



ggcattctgtatttaattggtaatctgagatggcagggaacagc



atcagcatggtgtgagggataggcattttttcattgtgtacagc



ttgtaaatcagtatttttaaaactcaaagttaatggcttgggca



tatttagaaaagagttgccgcacggacttgaaccctgtattcct



aaaatctaggatcttgttctgatggtctgcacaactggctgggg



gtgtccagccactgtccctcttgcctgggctccccagggcagtt



ctgtcagcctctccatttccattcctgttccagcaaaacccaac



tgatagcacagcagcatttcagcctgtctacctctgtgcccaca



tacctggatgtctaccagccagaaaggtggcttagatttggttc



ctgtgggtggattatggcccccagaacttccctgtgcttgctgg



gggtgtggagtggaaagagcaggaaatgggggaccctccgatac



tctatgggggtcctccaagtctctttgtgcaagttagggtaata



atcaatatggagctaagaaagagaaggggaactatgctttagaa



caggacactgtgccaggagcattgcagaaattatatggttttca



cgacagttctttttggtaggtactgttattatcctcagtttgca



gatgaggaaactgagacccagaaaggttaaataacttgctaggg



tcacacaagtcataactgacaaagcctgattcaaacccaggtct



ccctaacctttaaggtttctatgacgccagctctcctagggagt



ttgtcttcagatgtcttggctctaggtgtcaaaaaaagacttgg



tgtcaggcaggcataggttcaagtcccaactctgtcacttacca



actgtgactaggtgattgaactgaccatggaacctggtcacatg



caggagcaggatggtgaagggttcttgaaggcacttaggcagga



catttaggcaggagagaaaacctggaaacagaagagctgtctcc



aaaaatacccactggggaagcaggttgtcatgtgggccatgaat



gggacctgttctggtaaccaagcattgcttatgtgtccattaca



tttcataacacttccatcctactttacagggaacaaccaagact



ggggttaaatctcacagcctgcaagtggaagagaagaacttgaa



cccaggtccaacttttgcgccacagcaggctgcctcttggtcct



gacaggaagtcacaacttgggtctgagtactgatccctggctat



tttttggctgtgttaccttggacaagtcacttattcctcctccc



gtttcctcctatgtaaaatggaaataataatgttgaccctgggt



ctgagagagtggatttgaaagtacttagtgcatcacaaagcaca



gaacacacttccagtctcgtgattatgtacttatgtaactggtc



atcacccatcttgagaatgaatgcattggggaaagggccatcca



ctaggctgcgaagtttctgagggactccttcgggctggagaagg



atggccacaggagggaggagagattgccttatcctgcagtgatc



atgtcattgagaacagagccagattctttttttcctggcagggc



caacttgttttaacatctaaggactgagctatttgtgtctgtgc



cctttgtccaagcagtgtttcccaaagtgtagcccaagaaccat



ctccctcagagccaccaggaagtgctttaaattgcaggttccta



ggccacagcctgcacctgcagagtcagaatcatggaggttggga



cccaggcacctgcgtttctaacaaatgcctcgggtgattctgat



gcaattgaaagtttgagatccacagttctgagacaataacagaa



tggtttttctaacccctgcagccctgacttcctatcctagggaa



ggggccggctggagaggccaggacagagaaagcagatcccttct



ttttccaaggactctgtgtcttccataggcaac





HUMAN RPE65
11


PROMOTER
tacgtaatatttattgaagtttaatattgtgtttgtgatacaga



agtatttgctttaattctaaataaaaattttatgcttttattgc



tggtttaagaagatttggattatccttgtactttgaggagaagt



ttcttatttgaaatattttggaaacaggtcttttaatgtggaaa



gatagatattaatctcctcttctattactctccaagatccaaca



aaagtgattataccccccaaaatatgatggtagtatcttatact



accatcattttataggcatagggctcttagctgcaaataatgga



actaactctaataaagcagaacgcaaatattgtaaatattagag



agctaacaatctctgggatggctaaaggatggagcttggaggct



acccagccagtaacaatattccgggctccactgttgaatggaga



cactacaactgccttggatgggcagagatattatggatgctaag



ccccaggtgctaccattaggacttctaccactgtccctaacggg



tggagcccatcacatgcctatgccctcactgtaaggaaatgaag



ctactgttgtatatcttgggaagcacttggattaattgttatac



agttttgttgaagaagacccctagggtaagtagccataactgca



cactaaatttaaaattgttaatgagtttctcaaaaaaaatgtta



aggttgttagctggtatagtatatatcttgcctgttttccaagg



acttctttgggcagtaccttgtctgtgctggcaagcaactgaga



cttaatgaaagagtattggagatatgaatgaattgatgctgtat



actctcagagtgccaaacatataccaatggacaagaaggtgagg



cagagagcagacaggcattagtgacaagcaaagatatgcagaat



ttcattctcagcaaatcaaaagtcctcaacctggttggaagaat



attggcactgaatggtatcaataaggttgctagagagggttaga



ggtgcacaatgtgcttccataacattttatacttctccaatctt



agcactaatcaaacatggttgaatactttgtttactataactct



tacagagttataagatctgtgaagacagggacagggacaatacc



catctctgtctggttcataggtggtatgtaatagatatttttaa



aaataagtgagttaatgaatgagggtgagaatgaaggcacagag



gtattagggggaggtgggccccagagaatggtgccaaggtccag



tggggtgactgggatcagctcaggcctgacgctggccactccca



cctagctcctttctttctaatctgttctcattctccttgggaag



gattgaggtctctggaaaacagccaaacaactgttatgggaaca



gcaagcccaaataaagccaagcatcagggggatctgagagctga



aagcaacttctgttccccctccctcagctgaaggggtggggaag



ggctcccaaagccataactccttttaagggatttagaaggcata



aaaaggcccctggctgagaacttccttcttcattctgcagttggt





HUMAN VMD2
12


PROMOTER
tacgtaattctgtcattttactagggtgatgaaattcccaagca



acaccatccttttcagataagggcactgaggctgagagaggagc



tgaaacctacccggcgtcaccacacacaggtggcaaggctggga



ccagaaaccaggactgttgactgcagcccggtattcattctttc



catagcccacagggctgtcaaagaccccagggcctagtcagagg



ctcctccttcctggagagttcctggcacagaagttgaagctcag



cacagccccctaacccccaactctctctgcaaggcctcaggggt



cagaacactggtggagcagatcctttagcctctggattttaggg



ccatggtagagggggtgttgccctaaattccagccctggtctca



gcccaacaccctccaagaagaaattagaggggccatggccaggc



tgtgctagccgttgcttctgagcagattacaagaagggactaag



acaaggactcctttgtggaggtcctggcttagggagtcaagtga



cggcggctcagcactcacgtgggcagtgccagcctctaagagtg



ggcaggggcactggccacagagtcccagggagtcccaccagcct



agtcgccagacc





SYNUCLEIN
13


INTRONIC
gggccccggtgttatctcattcttttttctcctctgtaagttga


SEQUENCE AS
catgtgatgtgggaacaaaggggataaagtcattattttgtgct


STUFFER
aaaatcgtaattggagaggacctcctgttagctgggctttcttc


SEQUENCE
tatttattgtggtggttactggagttccttcttctagttttagg



atatatatatatattttttttttttctttccctgaagatataat



aatatatatacttctgaagattgagatttttaaattagttgtat



tgaaaactagctaatcagcaatttaaggctagcttgagacttat



gtcttgaatttgtttttgtaggctccaaaaccaaggagggagtg



gtgcatggtgtggcaacaggtaagctccattgtgcttatatcca



aagatgatatttaaagtatctagtgattagtgtggcccagtatt



caagattcctatgaaattgtaaaacaatcactgagcattctaag



aacatatcagtcttattgaaactgaattctttataaagtatttt



taaaaaggtaaatattgattataaataaaaaatatacttgccaa



gaataatgagggctttgaattgataagctatgtttaatttatag



taagtgggcatttaaatattctgaccaaaaatgtattgacaaac



tgctgacaaaaataaaatgtgaatattgccataattttaaaaaa



agagtaaaatttctgttgattacagtaaaatattttgaccttaa



attatgttgattacaatattcctttgataattcagagtgcattt



caggaaacacccttggacagtcagtaaattgtttattgtattta



tctttgtattgttatggtatagctatttgtacaaatattattgt



gcaattattacatttctgattatattattcatttggcctaaatt



taccaagaatttgaacaagtcaattaggtttacaatcaagaaat



atcaaaaatgatgaaaaggatgataatcatcatcagatgttgag



gaagatgacgatgagagtgccagaaatagagaaatcaaaggaga



accaaaatttaacaaattaaaagcccacagacttgctgtaatta



agttttctgttgtaagtactccacgtttcctggcagatgtggtg



aagcaaaagatataatcagaaatataatttatatgatcggaaag



cattaaacacaatagtgcctatacaaataaaatgttcctatcac



tgacttctaaaatggaaatgaggacaatgatatgggaatcttaa



tacagtgttgtggataggactaaaaacacaggagtcagatcttc



ttggttcaacttcctgcttactccttaccagctgtgtgtttttt



gcaaggttcttcacctctatgtgatttagcttcctcatctataa



aataattcagtgaattaatgtacacaaaacatctggaaaacaaa



agcaaacaatatgtattttataagtgttacttatagttttatag



tgaactttcttgtgcaacatttttacaactagtggagaaaaata



tttctttaaatgaatacttttgatttaaaaatcagagtgtaaaa



ataaaacagactcctttgaaactagttctgttagaagttaattg



tgcacctttaatgggctctgttgcaatccaacagagaagtagtt



aagtaagtggactatgatggcttctagggacctcctataaatat



gatattgtgaagcatgattataataagaactagataacagacag



gtggagactccactatctgaagagggtcaacctagatgaatggt



gttccatttagtagttgaggaagaacccatgaggtttagaaagc



agacaagcatgtggcaagttctggagtcagtggtaaaaattaaa



gaacccaactattactgtcacctaatgatctaatggagactgtg



gagatgggctgcatttttttaatcttctccagaatgccaaaatg



taaacacatatctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg



agagagagagagagagagagagagactgaagtttgtacaattag



acattttataaaatgttttctgaaggacagtggctcacaatctt



aagtttctaacattgtacaatgttgggagactttgtatacttta



ttttctctttagcatattaaggaatctgagatgtcctacagtaa



agaaatttgcattacatagttaaaatcagggttattcaaacttt



ttgattattgaaacctttcttcattagttactagggttgaatga



aactagtgttccacagaaaactatgggaaatgttgctaggcagt



aaggacatggtgatttcagcatgtgcaatatttacagcgattgc



acccatggaccaccctggcagtagtgaaataaccaaaaatgctg



tcataactagtatggctatgagaaacacattggg





RLBP1 INTRONIC
14


SEQUENCE AS
ATTCTCCAGGTTGAGCCAGACCAATTTGATGGTAGATTTAGCAA


STUFFER
ATAAAAATACAGGACACCCAGTTAAATGTGAATTTCCGATGAAC


SEQUENCE
AGCAAATACTTTTTTAGTATTAAAAAAGTTCACATTTAGGCTCA


(NT_010274.17)
CGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCAGATCA



CCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAA



CCCCATCTCCACTAAAAATACCAAAAATTAGCCAGGCGTGCTGG



TGGGCACCTGTAGTTCCAGCTACTCAGGAGGCTAAGGCAGGAGA



ATTGCTTGAACCTGGGAGGCAGAGGTTGCAGTGAGCTGAGATCG



CACCATTGCACTCTAGCCTGGGCGACAAGAACAAAACTCCATCT



CAAAAAAAAAAAAAAAAAAAAAGTTCACATTTAACTGGGCATTC



TGTATTTAATTGGTAATCTGAGATGGCAGGGAACAGCATCAGCA



TGGTGTGAGGGATAGGCATTTTTTCATTGTGTACAGCTTGTAAA



TCAGTATTTTTAAAACTCAAAGTTAATGGCTTGGGCATATTTAG



AAAAGAGTTGCCGCACGGACTTGAACCCTGTATTCCTAAAATCT



AGGATCTTGTTCTGATGGTCTGCACAACTGGCTGGGGGTGTCCA



GCCACTGTCCCTCTTGCCTGGGCTCCCCAGGGCAGTTCTGTCAG



CCTCTCCATTTCCATTCCTGTTCCAGCAAAACCCAACTGATAGC



ACAGCAGCATTTCAGCCTGTCTACCTCTGTGCCCACATACCTGG



ATGTCTACCAGCCAGAAAGGTGGCTTAGATTTGGTTCCTGTGGG



TGGATTATGGCCCCCAGAACTTCCCTGTGCTTGCTGGGGGTGTG



GAGTGGAAAGAGCAGGAAATGGGGGACCCTCCGATACTCTATGG



GGGTCCTCCAAGTCTCTTTGTGCAAGTTAGGGTAATAATCAATA



TGGAGCTAAGAAAGAGAAGGGGAACTATGCTTTAGAACAGGACA



CTGTGCCAGGAGCATTGCAGAAATTATATGGTTTTCACGACAGT



TCTTTTTGGTAGGTACTGTTATTATCCTCAGTTTGCAGATGAGG



AAACTGAGACCCAGAAAGGTTAAATAACTTGCTAGGGTCACACA



AGTCATAACTGACAAAGCCTGATTCAAACCCAGGTCTCCCTAAC



CTTTAAGGTTTCTATGACGCCAGCTCTCCTAGGGAGTTTGTCTT



CAGATGTCTTGGCTCTAGGTGTCAAAAAAAGACTTGGTGTCAGG



CAGGCATAGGTTCAAGTCCCAACTCTGTCACTTACCAACTGTGA



CTAGGTGATTGAACTGACCATGGAACCTGGTCACATGCAGGAGC



AGGATGGTGAAGGGTTCTTGAAGGCACTTAGGCAGGACATTTAG



GCAGGAGAGAAAACCTGGAAACAGAAGAGCTGTCTCCAAAAATA



CCCACTGGGGAAGCAGGTTGTCATGTGGGCCATGAATGGGACCT



GTTCTGG





AMP BACTERIAL
15


BACKBONE
ctgcctgcaggggcgcctgatgcggtattttctccttacgcatc



tgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacg



cgccctgtagcggcgcattaagcgcggcgggtgtggtggttacg



cgcagcgtgaccgctacacttgccagcgccttagcgcccgctcc



tttcgctttcttcccttcctttctcgccacgttcgccggctttc



cccgtcaagctctaaatcgggggctccctttagggttccgattt



agtgctttacggcacctcgaccccaaaaaacttgatttgggtga



tggttcacgtagtgggccatcgccctgatagacggtttttcgcc



ctttgacgttggagtccacgttctttaatagtggactcttgttc



caaactggaacaacactcaactctatctcgggctattcttttga



tttataagggattttgccgatttcggtctattggttaaaaaatg



agctgatttaacaaaaatttaacgcgaattttaacaaaatatta



acgtttacaattttatggtgcactctcagtacaatctgctctga



tgccgcatagttaagccagccccgacacccgccaacacccgctg



acgcgccctgacgggcttgtctgctcccggcatccgcttacaga



caagctgtgaccgtctccgggagctgcatgtgtcagaggttttc



accgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatac



gcctatttttataggttaatgtcatgataataatggtttcttag



acgtcaggtggcacttttcggggaaatgtgcgcggaacccctat



ttgtttatttttctaaatacattcaaatatgtatccgctcatga



gacaataaccctgataaatgcttcaataatattgaaaaaggaag



agtatgagtattcaacatttccgtgtcgcccttattcccttttt



tgcggcattttgccttcctgtttttgctcacccagaaacgctgg



tgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggt



tacatcgaactggatctcaacagcggtaagatccttgagagttt



tcgccccgaagaacgttttccaatgatgagcacttttaaagttc



tgctatgtggcgcggtattatcccgtattgacgccgggcaagag



caactcggtcgccgcatacactattctcagaatgacttggttga



gtactcaccagtcacagaaaagcatcttacggatggcatgacag



taagagaattatgcagtgctgccataaccatgagtgataacact



gcggccaacttacttctgacaacgatcggaggaccgaaggagct



aaccgcttttttgcacaacatgggggatcatgtaactcgccttg



atcgttgggaaccggagctgaatgaagccataccaaacgacgag



cgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaa



actattaactggcgaactacttactctagcttcccggcaacaat



taatagactggatggaggcggataaagttgcaggaccacttctg



cgctcggcccttccggctggctggtttattgctgataaatctgg



agccggtgagcgtgggtctcgcggtatcattgcagcactggggc



cagatggtaagccctcccgtatcgtagttatctacacgacgggg



agtcaggcaactatggatgaacgaaatagacagatcgctgagat



aggtgcctcactgattaagcattggtaactgtcagaccaagttt



actcatatatactttagattgatttaaaacttcatttttaattt



aaaaggatctaggtgaagatcctttttgataatctcatgaccaa



aatcccttaacgtgagttttcgttccactgagcgtcagaccccg



tagaaaagatcaaaggatcttcttgaaatcctttttttctgcgc



gtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggt



ggtttgtttgccggatcaagagctaccaactctttttccgaagg



taactggcttcagcagagcgcagataccaaatactgttcttcta



gtgtagccgtagttaggccaccacttcaagaactctgtagcacc



gcctacatacctcgctctgctaatcctgttaccagtggctgctg



ccagtggcgataagtcgtgtcttaccgggttggactcaagacga



tagttaccggataaggcgcagcggtcgggctgaacggggggttc



gtgcacacagcccagcttggagcgaacgacctacaccgaactga



gatacctacagcgtgagctatgagaaagcgccacgcttcccgaa



gggagaaaggcggacaggtatccggtaagcggcagggtcggaac



aggagagcgcacgagggagcttccagggggaaacgcctggtatc



tttatagtcctgtcgggtttcgccacctctgacttgagcgtcga



tttttgtgatgctcgtcaggggggcggagcctatggaaaaacgc



cagcaacgcggcctttttacggttcctggccttttgctggcctt



ttgctcacatgtcctgcaggcag





5′ ITR -
16


STRATAGENE
Ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgg



gcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgag



cgcgcagagagggagtggccaactccatcactaggggttcct





5′ ITR - NCBI
17


(AF043303)
Ttggccactccctctctgcgcgctcgctcgctcactgaggccgg



gcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcct



cagtgagcgagcgagcgcgcagagagggagtggccaactccatc



actaggggttcct





AAV2 CAPSID
18


CODING SEQUENCE
atggctgccgatggttatcttccagattggctcgaggacactct



ctctgaaggaataagacagtggtggaagctcaaacctggcccac



caccaccaaagcccgcagagcggcataaggacgacagcaggggt



cttgtgcttcctgggtacaagtacctcggacccttcaacggact



cgacaagggagagccggtcaacgaggcagacgccgcggccctcg



agcacgacaaagcctacgaccggcagctcgacagcggagacaac



ccgtacctcaagtacaaccacgccgacgcggagtttcaggagcg



ccttaaagaagatacgtcttttgggggcaacctcggacgagcag



tcttccaggcgaaaaagagggttcttgaacctctgggcctggtt



gaggaacctgttaagacggctccgggaaaaaagaggccggtaga



gcactctcctgtggagccagactcctcctcgggaaccggaaagg



cgggccagcagcctgcaagaaaaagattgaattttggtcagact



ggagacgcagactcagtacctgacccccagcctctcggacagcc



accagcagccccctctggtctgggaactaatacgatggctacag



gcagtggcgcaccaatggcagacaataacgagggcgccgacgga



gtgggtaattcctcgggaaattggcattgcgattccacatggat



gggcgacagagtcatcaccaccagcacccgaacctgggccctgc



ccacctacaacaaccacctctacaaacaaatttccagccaatca



ggagcctcgaacgacaatcactactttggctacagcaccccttg



ggggtattttgacttcaacagattccactgccacttttcaccac



gtgactggcaaagactcatcaacaacaactggggattccgaccc



aagagactcaacttcaagctctttaacattcaagtcaaagaggt



cacgcagaatgacggtacgacgacgattgccaataaccttacca



gcacggttcaggtgtttactgactcggagtaccagctcccgtac



gtcctcggctcggcgcatcaaggatgcctcccgccgttcccagc



agacgtcttcatggtgccacagtatggatacctcaccctgaaca



acgggagtcaggcagtaggacgctcttcattttactgcctggag



tactttccttctcagatgctgcgtaccggaaacaactttacctt



cagctacacttttgaggacgttcctttccacagcagctacgctc



acagccagagtctggaccgtctcatgaatcctctcatcgaccag



tacctgtattacttgagcagaacaaacactccaagtggaaccac



cacgcagtcaaggcttcagttttctcaggccggagcgagtgaca



ttcgggaccagtctaggaactggcttcctggaccctgttaccgc



cagcagcgagtatcaaagacatctgcggataacaacaacagtga



atactcgtggactggagctaccaagtaccacctcaatggcagag



actctctggtgaatccgggcccggccatggcaagccacaaggac



gatgaagaaaagttttttcctcagagcggggttctcatctttgg



gaagcaaggctcagagaaaacaaatgtggacattgaaaaggtca



tgattacagacgaagaggaaatcaggacaaccaatcccgtggct



acggagcagtatggttctgtatctaccaacctccagagaggcaa



cagacaagcagctaccgcagatgtcaacacacaaggcgttcttc



caggcatggtctggcaggacagagatgtgtaccttcaggggccc



atctgggcaaagattccacacacggacggacattttcacccctc



tcccctcatgggtggattcggacttaaacaccctcctccacaga



ttctcatcaagaacaccccggtacctgcgaatccttcgaccacc



ttcagtgcggcaaagtttgcttccttcatcacacagtactccac



gggacaggtcagcgtggagatcgagtgggagctgcagaaggaaa



acagcaaacgctggaatcccgaaattcagtacacttccaactac



aacaagtctgttaatgtggactttactgtggacactaatggcgt



gtattcagagcctcgccccattggcaccagatacctgactcgta



atctgtaa





AAV2 CAPSID
19


SEQUENCE (VP1)
maadgylpdwledtlsegirqwwklkpgppppkpaerhkddsrg



lvlpgykylgpfngldkgepvneadaaalehdkaydrqldsgdn



pylkynhadaefqerlkedtsfggnlgravfqakkrvleplglv



eepvktapgkkrpvehspvepdsssgtgkagqqparkrlnfgqt



gdadsvpdpqplgqppaapsglgtntmatgsgapmadnnegadg



vgnssgnwhcdstwmgdrvittstrtwalptynnhlykqissqs



gasndnhyfgystpwgyfdfnrfhchfsprdwqrlinnnwgfrp



krlnfklfniqvkevtqndgtttiannltstvqvftdseyqlpy



vlgsahqgclppfpadvfmvpqygyltlnngsqavgrssfycle



yfpsqmlrtgnnftfsytfedvpfhssyahsqsldrlmnplidq



ylyylsrtntpsgtttqsrlqfsqagasdirdqsrnwlpgpcyr



qqrvsktsadnnnseyswtgatkyhlngrdslvnpgpamashkd



deekffpqsgvlifgkqgsektnvdiekvmitdeeeirttnpva



teqygsvstnlqrgnrqaatadvntqgvlpgmvwqdrdvylqgp



iwakiphtdghfhpsplmggfglkhpppqilikntpvpanpstt



fsaakfasfitqystgqvsveiewelqkenskrwnpeiqytsny



nksvnvdftvdtngvyseprpigtryltrnl





AAV2 CAPSID
68


SEQUENCE (VP2)
mapgkkrpvehspvepdsssgtgkagqqparkrlnfgqtgdads



vpdpqplgqppaapsglgtntmatgsgapmadnnegadgvgnss



gnwhcdstwmgdrvittstrtwalptynnhlykqissqsgasnd



nhyfgystpwgyfdfnrfhchfsprdwqrlinnnwgfrpkrlnf



klfniqvkevtqndgtttiannltstvqvftdseyqlpyvlgsa



hqgclppfpadvfmvpqygyltlnngsqavgrssfycleyfpsq



mlrtgnnftfsytfedvpfhssyahsqsldrlmnplidqylyyl



srtntpsgtttqsrlqfsqagasdirdqsrnwlpgpcyrqqrvs



ktsadnnnseyswtgatkyhlngrdslvnpgpamashkddeekf



fpqsgvlifgkqgsektnvdiekvmitdeeeirttnpvateqyg



svstnlqrgnrqaatadvntqgvlpgmvwqdrdvylqgpiwaki



phtdghfhpsplmggfglkhpppqilikntpvpanpsttfsaak



fasfitqystgqvsveiewelqkenskrwnpeiqytsnynksvn



vdftvdtngvyseprpigtryltrnl





AAV2 CAPSID
69


SEQUENCE (VP3)
matgsgapmadnnegadgvgnssgnwhcdstwmgdrvittstrt



walptynnhlykqissqsgasndnhyfgystpwgyfdfnrfhch



fsprdwqrlinnnwgfrpkrlnfklfniqvkevtqndgtttian



nltstvqvftdseyqlpyvlgsahqgclppfpadvfmvpqygyl



tlnngsqavgrssfycleyfpsqmlrtgnnftfsytfedvpfhs



syahsqsldrlmnplidqylyylsrtntpsgtttqsrlqfsqag



asdirdqsrnwlpgpcyrqqrvsktsadnnnseyswtgatkyhl



ngrdslvnpgpamashkddeekffpqsgvlifgkqgsektnvdi



ekvmitdeeeirttnpvateqygsvstnlqrgnrqaatadvntq



gvlpgmvwqdrdvylqgpiwakiphtdghfhpsplmggfglkhp



ppqilikntpvpanpsttfsaakfasfitqystgqvsveiewel



qkenskrwnpeiqytsnynksvnvdftvdtngvyseprpigtry



ltrnl





AAV8 CAPSID
20


CODING SEQUENCE
atggctgccgatggttatcttccagattggctcgaggacaacct



ctctgagggcattcgcgagtggtgggcgctgaaacctggagccc



cgaagcccaaagccaaccagcaaaagcaggacgacggccggggt



ctggtgcttcctggctacaagtacctcggacccttcaacggact



cgacaagggggagcccgtcaacgcggcggacgcagcggccctcg



agcacgacaaggcctacgaccagcagctgcaggcgggtgacaat



ccgtacctgcggtataaccacgccgacgccgagtttcaggagcg



tctgcaagaagatacgtcttttgggggcaacctcgggcgagcag



tcttccaggccaagaagcgggttctcgaacctctcggtctggtt



gaggaaggcgctaagacggctcctggaaagaagagaccggtaga



gccatcaccccagcgttctccagactcctctacgggcatcggca



agaaaggccaacagcccgccagaaaaagactcaattttggtcag



actggcgactcagagtcagttccagaccctcaacctctcggaga



acctccagcagcgccctctggtgtgggacctaatacaatggctg



caggcggtggcgcaccaatggcagacaataacgaaggcgccgac



ggagtgggtagttcctcgggaaattggcattgcgattccacatg



gctgggcgacagagtcatcaccaccagcacccgaacctgggccc



tgcccacctacaacaaccacctctacaagcaaatctccaacggg



acatcgggaggagccaccaacgacaacacctacttcggctacag



caccccctgggggtattttgactttaacagattccactgccact



tttcaccacgtgactggcagcgactcatcaacaacaactgggga



ttccggcccaagagactcagcttcaagctcttcaacatccaggt



caaggaggtcacgcagaatgaaggcaccaagaccatcgccaata



acctcaccagcaccatccaggtgtttacggactcggagtaccag



ctgccgtacgttctcggctctgcccaccagggctgcctgcctcc



gttcccggcggacgtgttcatgattccccagtacggctacctaa



cactcaacaacggtagtcaggccgtgggacgctcctccttctac



tgcctggaatactttccttcgcagatgctgagaaccggcaacaa



cttccagtttacttacaccttcgaggacgtgcctttccacagca



gctacgcccacagccagagcttggaccggctgatgaatcctctg



attgaccagtacctgtactacttgtctcggactcaaacaacagg



aggcacggcaaatacgcagactctgggcttcagccaaggtgggc



ctaatacaatggccaatcaggcaaagaactggctgccaggaccc



tgttaccgccaacaacgcgtctcaacgacaaccgggcaaaacaa



caatagcaactttgcctggactgctgggaccaaataccatctga



atggaagaaattcattggctaatcctggcatcgctatggcaaca



cacaaagacgacgaggagcgtttttttcccagtaacgggatcct



gatttttggcaaacaaaatgctgccagagacaatgcggattaca



gcgatgtcatgctcaccagcgaggaagaaatcaaaaccactaac



cctgtggctacagaggaatacggtatcgtggcagataacttgca



gcagcaaaacacggctcctcaaattggaactgtcaacagccagg



gggccttacccggtatggtctggcagaaccgggacgtgtacctg



cagggtcccatctgggccaagattcctcacacggacggcaactt



ccacccgtctccgctgatgggcggctttggcctgaaacatcctc



cgcctcagatcctgatcaagaacacgcctgtacctgcggatcct



ccgaccaccttcaaccagtcaaagctgaactctttcatcacgca



atacagcaccggacaggtcagcgtggaaattgaatgggagctgc



agaaggaaaacagcaagcgctggaaccccgagatccagtacacc



tccaactactacaaatctacaagtgtggactttgctgttaatac



agaaggcgtgtactctgaaccccgccccattggcacccgttacc



tcacccgtaatctgtaa





AAV8 CAPSID
21


SEQUENCE (VP1)
maadgylpdwlednlsegirewwalkpgapkpkanqqkqddgrg



lvlpgykylgpfngldkgepvnaadaaalehdkaydqqlqagdn



pylrynhadaefqerlqedtsfggnlgravfqakkrvleplglv



eegaktapgkkrpvepspqrspdsstgigkkgqqparkrlnfgq



tgdsesvpdpqplgeppaapsgvgpntmaagggapmadnnegad



gvgsssgnwhcdstwlgdrvittstrtwalptynnhlykqisng



tsggatndntyfgystpwgyfdfnrfhchfsprdwqrlinnnwg



frpkrlsfklfniqvkevtqnegtktiannltstiqvftdseyq



lpyvlgsahqgclppfpadvfmipqygyltlnngsqavgrssfy



cleyfpsqmlrtgnnfqftytfedvpfhssyahsqsldrlmnpl



idqylyylsrtqttggtantqtlgfsqggpntmanqaknwlpgp



cyrqqrvstttgqnnnsnfawtagtkyhlngrnslanpgiamat



hkddeerffpsngilifgkqnaardnadysdvmltseeeikttn



pvateeygivadnlqqqntapqigtvnsqgalpgmvwqnrdvyl



qgpiwakiphtdgnfhpsplmggfglkhpppqilikntpvpadp



pttfnqsklnsfitqystgqvsveiewelqkenskrwnpeiqyt



snyykstsvdfavntegvyseprpigtryltrnl





AAV8 CAPSID
70


SEQUENCE (VP2)
mapgkkrpvepspqrspdsstgigkkgqqparkrlnfgqtgdse



svpdpqplgeppaapsgvgpntmaagggapmadnnegadgvgss



sgnwhcdstwlgdrvittstrtwalptynnhlykqisngtsgga



tndntyfgystpwgyfdfnrfhchfsprdwqrlinnnwgfrpkr



lsfklfniqvkevtqnegtktiannltstiqvftdseyqlpyvl



gsahqgclppfpadvfmipqygyltlnngsqavgrssfycleyf



psqmlrtgnnfqftytfedvpfhssyahsqsldrlmnplidqyl



yylsrtqttggtantqtlgfsqggpntmanqaknwlpgpcyrqq



rvstttgqnnnsnfawtagtkyhlngrnslanpgiamathkdde



erffpsngilifgkqnaardnadysdvmltseeeikttnpvate



eygivadnlqqqntapqigtvnsqgalpgmvwqnrdvylqgpiw



akiphtdgnfhpsplmggfglkhpppqilikntpvpadppttfn



qsklnsfitqystgqvsveiewelqkenskrwnpeiqytsnyyk



stsvdfavntegvyseprpigtryltrnl





AAV8 CAPSID
71


SEQUENCE (VP3)
maagggapmadnnegadgvgsssgnwhcdstwlgdrvittstrt



walptynnhlykqisngtsggatndntyfgystpwgyfdfnrfh



chfsprdwqrlinnnwgfrpkrlsfklfniqvkevtqnegtkt



annltstiqvftdseyqlpyvlgsahqgclppfpadvfmipqyg



yltlnngsqavgrssfycleyfpsqmlrtgnnfqftytfedvpf



hssyahsqsldrlmnplidqylyylsrtqttggtantqtlgfsq



ggpntmanqaknwlpgpcyrqqrvstttgqnnnsnfawtagtky



hlngrnslanpgiamathkddeerffpsngilifgkqnaardna



dysdvmltseeeikttnpvateeygivadnlqqqntapqigtvn



sqgalpgmvwqnrdvylqgpiwakiphtdgnfhpsplmggfglk



hpppqilikntpvpadppttfnqsklnsfitqystgqvsveiew



elqkenskrwnpeiqytsnyykstsvdfavntegvyseprpigt



ryltrnl





CVM ENHANCER
22


AND CBA
ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAG


PROMOTER
CCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCC


(GENBANK
CGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA


ACCESSION
ATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG


DD215332 FROM
ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG


BP 1-BP 1616)
TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC



AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC



CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA



TCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACT



CTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATT



TATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGG



GGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGC



GGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGC



GCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGC



CCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGAC



GCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGC



CCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGG



GCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT



AATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAG



GGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGG



TGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCG



CGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTT



TGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGG



TGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCG



TGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCG



TCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGC



TGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTG



GCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGG



GTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGG



GGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCG



CGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGG



GCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATC



TGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGC



GGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGT



GCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGG



CTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGG



CGGGGTTCGGCTTCTGGCGTGTGACCGGCGGC





REVERSE
23


COMPLEMENT OF
CCAGAACAGGTCCCATTCATGGCCCACATGACAACCTGCTTCCC


RLBP1 INTRONIC
CAGTGGGTATTTTTGGAGACAGCTCTTCTGTTTCCAGGTTTTCT


SEQUENCE AS
CTCCTGCCTAAATGTCCTGCCTAAGTGCCTTCAAGAACCCTTCA


STUFFER
CCATCCTGCTCCTGCATGTGACCAGGTTCCATGGTCAGTTCAAT


SEQUENCE
CACCTAGTCACAGTTGGTAAGTGACAGAGTTGGGACTTGAACCT


(NT_010274.17)
ATGCCTGCCTGACACCAAGTCTTTTTTTGACACCTAGAGCCAAG



ACATCTGAAGACAAACTCCCTAGGAGAGCTGGCGTCATAGAAAC



CTTAAAGGTTAGGGAGACCTGGGTTTGAATCAGGCTTTGTCAGT



TATGACTTGTGTGACCCTAGCAAGTTATTTAACCTTTCTGGGTC



TCAGTTTCCTCATCTGCAAACTGAGGATAATAACAGTACCTACC



AAAAAGAACTGTCGTGAAAACCATATAATTTCTGCAATGCTCCT



GGCACAGTGTCCTGTTCTAAAGCATAGTTCCCCTTCTCTTTCTT



AGCTCCATATTGATTATTACCCTAACTTGCACAAAGAGACTTGG



AGGACCCCCATAGAGTATCGGAGGGTCCCCCATTTCCTGCTCTT



TCCACTCCACACCCCCAGCAAGCACAGGGAAGTTCTGGGGGCCA



TAATCCACCCACAGGAACCAAATCTAAGCCACCTTTCTGGCTGG



TAGACATCCAGGTATGTGGGCACAGAGGTAGACAGGCTGAAATG



CTGCTGTGCTATCAGTTGGGTTTTGCTGGAACAGGAATGGAAAT



GGAGAGGCTGACAGAACTGCCCTGGGGAGCCCAGGCAAGAGGGA



CAGTGGCTGGACACCCCCAGCCAGTTGTGCAGACCATCAGAACA



AGATCCTAGATTTTAGGAATACAGGGTTCAAGTCCGTGCGGCAA



CTCTTTTCTAAATATGCCCAAGCCATTAACTTTGAGTTTTAAAA



ATACTGATTTACAAGCTGTACACAATGAAAAAATGCCTATCCCT



CACACCATGCTGATGCTGTTCCCTGCCATCTCAGATTACCAATT



AAATACAGAATGCCCAGTTAAATGTGAACTTTTTTTTTTTTTTT



TTTTTTGAGATGGAGTTTTGTTCTTGTCGCCCAGGCTAGAGTGC



AATGGTGCGATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTC



AAGCAATTCTCCTGCCTTAGCCTCCTGAGTAGCTGGAACTACAG



GTGCCCACCAGCACGCCTGGCTAATTTTTGGTATTTTTAGTGGA



GATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGA



CCTCAGGTGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGGATT



ACAGGCGTGAGCCTAAATGTGAACTTTTTTAATACTAAAAAAGT



ATTTGCTGTTCATCGGAAATTCACATTTAACTGGGTGTCCTGTA



TTTTTATTTGCTAAATCTACCATCAAATTGGTCTGGCTCAACCT



GGAGAAT





EGFP SEQUENCE
24



ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCAT



CCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG



TGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC



CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC



CACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC



GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCC



ATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGA



CGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG



ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAG



GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA



CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCA



TCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC



GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGA



CGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGT



CCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC



CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGA



CGAGCTGTACAAGTAA





GFP AMINO ACID
25


SEQUENCE
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT



LKFICT



TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYV



QERTIF



FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY



NYNSHN



VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPV



LLPDNH



YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK





SC5′ITR
36



CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGG



GCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAG



CGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT






MACACA MULATTA

37


(RHESUS MONKEY)
ATGTCAGAAGGGGTGGGCACGTTCCGCATGGTACCTGAAGAGGA


RLBP1 CDS
ACAGGAGCTCCGTGCCCAACTGGAGCAGCTCACAACCAAGGACC


XM_001091538
ATGGACCTGTCTTTGGCCCGTGCAGCCAGCTGCCCCGCCACACC



TTGCAGAAGGCCAAAGATGAGCTGAATGAGAGAGAGGAGACCCG



GGAGGAGGCAGTGCGAGAGCTGCAGGAGATGGTGCAGGCGCAGG



CGGCCTCGGGGGAGGAGCTGGCCGTGGCCGTGGCGGAGAGGGTG



CAAGAGAAGGACAGCGGCTTCTTCCTGCGCTTCATCCGCGCGCG



AAAGTTCAACGTGGGCCGTGCCTATGAGCTGCTCAGAGGCTATG



TGAATTTCCGGCTGCAGTACCCTGAGCTCTTTGACAGCCTGTCC



CCAGAGGCTGTCCGCTGTACCATTGAAGCTGGCTACCCTGGTGT



CCTCTCTAGTCGGGACAAGTATGGCCGAGTGGTCATGCTCTTCA



ACATTGAGAACTGGCAAAGTCAAGAAATCACCTTCGATGAGATC



TTGCAGGCATATTGCTTCATCCTGGAGAAGCTGCTGGAGAATGA



GGAAACTCAAATTAATGGATTCTGCATCATTGAGAACTTCAAGG



GCTTTACCATGCAGCAGGCTGCTAGTCTCCGCACTTCAGATCTC



AGGAAGATGGTGGACATGCTCCAGGATTCCTTCCCAGCCCGGTT



CAAAGCCATCCACTTCATCCACCAGCCATGGTACTTCACCACGA



CCTACAATGTGGTCAAGCCCTTCTTGAAGAGCAAGCTGCTTGAG



AGGGTCTTTGTCCACGGGGAGGACCTCTCTGGTTTCTACCAGGA



GATTGATGAGAACATCCTGCCCTCTGACTTTGGGGGCACGCTGC



CCAAGTATGATGGCAAAGCTGTTGCTGAGCAGCTCTTTGGCCCC



CGGGCCCAAGCTGAGAACACAGCCTTCTGA






MACACA MULATTA

38


(RHESUS MONKEY)
MSEGVGTFRMVPEEEQELRAQLEQLTTKDHGPVFGPCSQLPRHT


RLBP1 GENE
LQKAKDELNEREETREEAVRELQEMVQAQAASGEELAVAVAERV


PRODUCT
QEKDSGFFLRFIRARKFNVGRAYELLRGYVNFRLQYPELFDSLS


(CELLULAR
PEAVRCTIEAGYPGVLSSRDKYGRVVMLFNIENWQSQEITFDEI


RETINALDEHYDE
LQAYCFILEKLLENEETQINGFCIIENFKGFTMQQAASLRTSDL


BINDING PROTEIN -
RKMVDMLQDSFPARFKAIHFIHQPWYFTTTYNVVKPFLKSKLLE


CRALBP)
RVFVHGEDLSGFYQEIDENILPSDFGGTLPKYDGKAVAEQLFGP



RAQAENTAF






BOS TAURUS

39


RLBP1 CDS
ATGTCAGAGGGGGCGGGCACGTTCCGCATGGTCCCTGAAGAGGA


NM_174451
ACAGGAGCTCCGTGCCCAACTGGAGAGGCTTACGACCAAAGACC



ATGGACCTGTCTTTGGCCCGTGCAGCCAGCTGCCCCGCCACACC



TTGCAGAAGGCCAAGGACGAGCTGAATGAAAAGGAAGAGACCCG



GGAAGAGGCAGTGCGGGAGCTACAGGAGCTGGTGCAGGCGGAGG



CCGCCTCGGGGCAGGAGCTGGCCGTGGCCGTGGCGGAGAGGGTG



CAGGGAAAAGACAGTGCCTTCTTCCTGCGCTTCATCCGCGCGCG



CAAGTTCCACGTGGGGCGCGCCTACGAGCTGCTCAGAGGCTACG



TGAACTTCCGGCTGCAGTACCCAGAGCTCTTCGACAGCCTGTCC



CCAGAGGCTGTCCGCTGCACCGTTGAGGCTGGCTACCCTGGTGT



CCTCTCCACGCGGGACAAGTATGGCCGAGTGGTCATGCTCTTCA



ATATTGAGAACTGGGACTCTGAAGAAATCACCTTTGATGAGATC



TTGCAGGCATACTGCGTCATCCTGGAGAAGCTACTGGAGAATGA



GGAGACTCAAATTAATGGCTTTTGCATCATTGAGAACTTCAAGG



GCTTCACCATGCAGCAGGCTGCCGGACTTCGGCCTTCCGATC



TCAGAAAGATGGTGGACATGCTCCAGGATTCCTTCCCAGCTCGG



TTCAAAGCCATCCACTTCATCTACCAGCCCTGGTACTTCACCAC



CACCTACAACGTGGTCAAGCCCTTCTTGAAGAGCAAATTGCTCC



AGAGGGTATTTGTCCATGGAGAAGACCTCTCCAGCTTCTACCAG



GAGTTTGACGAGGACATCCTGCCCTCCGACTTTGGGGGTACACT



GCCCAAGTATGATGGCAAGGCCGTTGCTGAGCAGCTCTTTGGTC



CTCGGGACCAAACTGAGAACACAGCCTTCTGA






BOS TAURUS

40


RLBP1 GENE
MSEGAGTFRMVPEEEQELRAQLERLTTKDHGPVFGPCSQLPRHT


PRODUCT
LQKAKDELNEKEETREEAVRELQELVQAEAASGQELAVAVAERV


(CELLULAR
QGKDSAFFLRFIRARKFHVGRAYELLRGYVNFRLQYPELFDSLS


RETINALDEHYDE
PEAVRCTVEAGYPGVLSTRDKYGRVVMLFNIENWDSEEITFDEI


BINDING PROTEIN -
LQAYCVILEKLLENEETQINGFCIIENFKGFTMQQAAGLRPSDL


CRALBP)
RKMVDMLQDSFPARFKAIHFIYQPWYFTTTYNVVKPFLKSKLLQ



RVFVHGEDLSSFYQEFDEDILPSDFGGTLPKYDGKAVAEQLFGP



RDQTENTAF






CANIS LUPUS

41



FAMILIARIS

ATGTCAGAAGGCGTGGGCACATTCCGTGTGGTCCCTGAAGAGGA


RLBP1 CDS
ACAGGAGCTCCGTGCCCAGCTGGAGCGGCTTACAACCAAGGACC


XM_549634
ATGGGCCTGTCTTTGGCCCTTGCAGCCAGCTCCCTCGTCATACC



TTACAGAAGGCCAAGGACGAGCTGAACGAGAGGGAGGAGACCCG



GGAGGAGGTGGTGCGAGAGCTGCAGGAGCTGGTGCAGGCACAGG



CTGCCACCGGGCAGGAGCTGGCCAGGGCGGTGGCTGAGAGGGTG



CAGGGAAGGGACAGTGCCTTCTTCCTGCGCTTCATCCGCGCGCG



GAAGTTCCATGTGGGGCGTGCCTACGAGCTGCTTCGAGGCTACG



TGAACTTCCGGCTGCAGTACCCAGAGCTCTTCGACAGCCTGTCC



CTGGAGGCTGTCCGTTGCACCGTCGAGGCCGGCTATCCTGGGGT



CCTCCCCAGTCGGGACAAGTATGGCCGAGTGGTCATGCTCTTCA



ACATCGAGAACTGGGACTCCGAAGAAATCACCTTCGATGAGATC



TTGCAGGCATATTGTTTCATCCTGGAGAAGCTACTAGAGAATGA



GGAAACTCAAATTAATGGCTTCTGCATTATTGAGAACTTTAAGG



GCTTTACCATGCAGCAGGCTGCTGGACTTCGGGCTTCCGATCTC



AGGAAGATGGTGGACATGCTCCAGGATTCCTTCCCAGCGCGGTT



CAAAGCCATCCACTTCATTCACCAACCATGGTACTTCACCACCA



CCTACAACATGGTCAAGCCCCTCCTGAAGAACAAGCTGCTCCAA



AGAGTCTTTGTCCATGGAGATGACCTCTCTGGCTTCTTCCAGGA



GATTGATGAAGACATACTGCCCGCTGACTTTGGGGGCACACTGC



CCAAGTATGATGGCAAGGTGGTTGCTGAGCAGCTCTTTGGCCCC



CGGGCCCAAGCTGAGAACACAGCCTTCTGA






CANIS LUPUS

42



FAMILIARIS

MSEGVGTFRVVPEEEQELRAQLERLTTKDHGPVFGPCSQLPRHT


RLBP1 GENE
LQKAKDELNEREETREEVVRELQELVQAQAATGQELARAVAERV


PRODUCT
QGRDSAFFLRFIRARKFHVGRAYELLRGYVNFRLQYPELFDSLS


(CELLULAR
LEAVRCTVEAGYPGVLPSRDKYGRVVMLFNIENWDSEEITFDEI


RETINALDEHYDE
LQAYCFILEKLLENEETQINGFCIIENFKGFTMQQAAGLRASDL


BINDING PROTEIN -
RKMVDMLQDSFPARFKAIHFIHQPWYFTTTYNMVKPLLKNKLLQ


CRALBP)
RVFVHGDDLSGFFQEIDEDILPADFGGTLPKYDGKVVAEQLFGP



RAQAENTAF






RATTUS

43



NORVEGICUS

ATGTCAGAGGGGGTGGGCACATTCCGAATGGTCCCTGAAGAGGA


RLBP1 CDS
GCAGGAGCTCCGGGCACAGCTAGAACAGCTCACAACCAAGGATC


NM_001106274.1
ATGGTCCTGTCTTTGGCCCATGCAGCCAGCTGCCCCGCCACACT



TTGCAGAAGGCTAAGGATGAGCTGAATGAAAGGGAGGAAACCCG



GGATGAGGCGGTGAGGGAGCTACAGGAGCTGGTCCAGGCACAGG



CAGCTTCTGGGGAAGAGTTGGCCGTGGCAGTGGCTGAGAGGGTG



CAGGCAAGAGACAGCGCCTTCCTCCTGCGCTTCATCCGTGCCCG



AAAGTTTGATGTGGGCCGGGCTTATGAGCTGCTCAAAGGCTATG



TGAACTTCCGGCTCCAGTACCCTGAACTCTTCGATAGCCTATC



TATGGAGGCTCTCCGCTGCACTATCGAGGCCGGTTACCCTGGTG



TCCTTTCCAGTCGGGACAAGTATGGTCGAGTGGTTATGCTCTTC



AACATTGAAAACTGGCACTGTGAAGAAGTCACCTTTGATGAGAT



CTTACAGGCATATTGTTTCATTCTGGAGAAACTGCTGGAGAACG



AGGAAACCCAAATCAACGGCTTCTGTATTGTGGAGAACTTCAAG



GGCTTCACCATGCAGCAGGCCGCGGGACTCCGCCCCTCCGATCT



CAAGAAGATGGTGGACATGCTCCAGGATTCATTCCCAGCCAGGT



TCAAAGCTATCCACTTCATCCACCAACCATGGTACTTCACCACC



ACTTACAATGTGGTCAAGCCCTTCTTGAAGAACAAGTTGCTACA



GAGGGTCTTCGTTCATGGAGATGACCTGGACGGCTTCTTCCAGG



AGATTGATGAGAATATCTTGCCTGCTGACTTTGGGGGTACACTG



CCCAAGTATGACGGCAAAGTTGTCGCTGAGCAGCTCTTCGGTCC



CCGGGTTGAGGTTGAGAACACAGCCTTGTGA






RATTUS

44



NORVEGICUS

MSEGVGTFRMVPEEEQELRAQLEQLTTKDHGPVFGPCSQLPRHT


RLBP1 GENE
LQKAKDELNEREETRDEAVRELQELVQAQAASGEELAVAVAERV


PRODUCT
QARDSAFLLRFIRARKFDVGRAYELLKGYVNFRLQYPELFDSLS


(CELLULAR
MEALRCTIEAGYPGVLSSRDKYGRVVMLFNIENWHCEEVTFDEI


RETINALDEHYDE
LQAYCFILEKLLENEETQINGFCIVENFKGFTMQQAAGLRPS


BINDING PROTEIN -
DLKKMVDMLQDSFPARFKAIHFIHQPWYFTTTYNVVKPFLKNKL


CRALBP)
LQRVFVHGDDLDGFFQEIDENILPADFGGTLPKYDGKVVAEQLF



GPRVEVENTAL






MUS MUSCULUS

45


RLBP1 CDS
ATGTCAGACGGGGTGGGCACTTTCCGCATGGTTCCTGAAGAGGA


NM_020599.2
GCAGGAGCTCCGAGCACAACTGGAGCAGCTCACAACCAAGGATC



ATGGTCCTGTCTTTGGCCCATGCAGCCAGCTGCCCCGCCACACT



TTGCAGAAGGCCAAGGATGAGCTGAATGAAAAGGAGGAGACCCG



GGAGGAAGCGGTGAGGGAGCTACAGGAGCTGGTACAGGCACAGG



CAGCTTCTGGCGAGGAATTGGCCCTGGCAGTGGCTGAGAGGGTG



CAGGCAAGAGACAGCGCCTTCCTCCTGCGCTTCATCCGTGCCCG



CAAGTTCGATGTGGGTCGTGCTTATGAGCTGCTCAAAGGCTATG



TGAACTTCCGCCTCCAGTACCCTGAACTCTTCGATAGTCTCTCC



ATGGAGGCTCTCCGCTGCACTATCGAGGCCGGATACCCTGGTGT



CCTTTCCAGTCGGGACAAGTATGGTCGAGTGGTTATGCTCTTCA



ACATCGAAAACTGGCACTGTGAAGAAGTGACCTTTGATGAGATC



TTACAGGCATATTGTTTCATTTTGGAGAAACTGCTGGAAAATGA



GGAAACCCAAATCAACGGCTTCTGTATTGTTGAGAACTTCAAGG



GCTTCACCATGCAGCAGGCAGCAGGGCTCCGCCCCTCGGATCTC



AAGAAGATGGTGGACATGCTCCAGGATTCATTCCCAGCCAGGTT



CAAAGCTATCCACTTCATCCACCAGCCATGGTACTTCACCACCA



CCTATAATGTGGTCAAGCCCTTCTTGAAGAACAAGCTGCTACAG



AGGGTCTTTGTTCACGGAGATGACCTGGATGGCTTCTTCCAGGA



GATTGATGAGAACATCCTGCCTGCTGACTTTGGGGGTACACTGC



CCAAGTACGACGGCAAAGTTGTTGCTGAGCAGCTCTTTGGTCCC



CGGGCTGAAGTTGAGAACACAGCCTTATGA






MUS MUSCULUS

46


RLBP1 GENE
MSDGVGTFRMVPEEEQELRAQLEQLTTKDHGPVFGPCSQLPRHT


PRODUCT
LQKAKDELNEKEETREEAVRELQELVQAQAASGEELALAVAERV


(CELLULAR
QARDSAFLLRFIRARKFDVGRAYELLKGYVNFRLQYPELFDSLS


RETINALDEHYDE
MEALRCTIEAGYPGVLSSRDKYGRVVMLFNIENWHCEEVTFDEI


BINDING PROTEIN -
LQAYCFILEKLLENEETQINGFCIVENFKGFTMQQAAGLRPSDL


CRALBP)
KKMVDMLQDSFPARFKAIHFIHQPWYFTTTYNVVKPFLKNKLLQ



RVFVHGDDLDGFFQEIDENILPADFGGTLPKYDGKVVAEQLFGP



RAEVENTAL






GALLUS GALLUS

47


RLBP1 CDS
ATGTCTGCTGTTACGGGCACCTTCCGCATTGTCTCGGAAGAGGA


NM_001024694.1
GCAGGCGCTGCGCACCAAACTGGAGCGCCTCACCACCAAGGACC



ACGGCCCTGTTTTTGGGAGGTGCCAGCAGATCCCCCCTCACACC



CTGCAGAAGGCAAAAGATGAGCTGAATGAGACGGAGGAGCAGAG



GGAGGCAGCGGTCAAAGCGCTGCGGGAGCTGGTGCAGGAGCGGG



CCGGCAGCGAGGATGTCTGCAAGGCAGTGGCAGAGAAGATGCAG



GGGAAGGACGATTCCTTCTTCCTCCGCTTCATCCGTGCCCGCAA



GTTTGACGTGCACAGGGCCTACGACCTGCTGAAAGGCTATGTGA



ACTTTCGCCAGCAATACCCTGAACTCTTTGACAACCTGACCCCC



GAGGCCGTGCGCAGCACCATCGAGGCGGGCTACCCCGGCATCCT



GGCCAGCAGGGACAAATACGGGCGGGTAGTGATGCTCTTCAACA



TCGAGAACTGGGACTACGAGGAGATCACCTTTGATGAGATCCTT



CGTGCCTACTGCGTTATCTTGGAGAAGCTGCTGGAAAACGAAGA



GACCCAGATCAATGGGTTCTGCATCATTGAGAACTTCAAGGGCT



TCACCATGCAGCAGGCATCAGGGATCAAACCCTCCGAGCTCAAG



AAGATGGTGGACATGCTACAGGACTCCTTCCCAGCGCGGTTCAA



AGCTGTCCACTTCATCCACCAGCCCTGGTACTTCACCACTACCT



ACAACGTGGTCAAACCGTTCCTGAAGAGCAAGCTGCTGGAGAGG



GTGTTTGTGCACGGCGAGGAGCTGGAGTCCTTCTACCAGGAG



ATCGATGCTGACATACTGCCAGCAGACTTCGGTGGCAACCTGCC



CAAGTACGACGGCAAAGCAACTGCAGAGCAGCTCTTTGGGCCCC



GCATTGAGGCTGAAGACACGGCACTTTAA






GALLUS GALLUS

48


RLBP1 GENE
MSAVTGTFRIVSEEEQALRTKLERLTTKDHGPVFGRCQQIPPHT


PRODUCT
LQKAKDELNETEEQREAAVKALRELVQERAGSEDVCKAVAEKMQ


(CELLULAR
GKDDSFFLRFIRARKFDVHRAYDLLKGYVNFRQQYPELFDNLTP


RETINALDEHYDE
EAVRSTIEAGYPGILASRDKYGRVVMLFNIENWDYEEITFDEIL


BINDING PROTEIN -
RAYCVILEKLLENEETQINGFCIIENFKGFTMQQASGIKPSELK


CRALBP)
KMVDMLQDSFPARFKAVHFIHQPWYFTTTYNVVKPFLKSKLLER


NP_001019865.1
VFVHGEELESFYQEIDADILPADFGGNLPKYDGKATAEQLFGPR



IEAEDTAL





KAN-R BACTERIAL
49


BACKBONE
CTGCCTGCAGGGTTCCATCCCAATGGCGCGTCAATTCACTGGCC



GTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCA



ACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTA



ATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGC



AGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTAC



GCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTA



CAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCG



CCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGC



ATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGT



GTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAG



GGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT



AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGC



GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG



TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATA



TTGAAAAAGGAAGAGTATGAGCCATATTCAACGGGAAACGTCTT



GCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATAT



GGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGAC



AATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTC



TGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAG



ATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGAC



CATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCA



CCACTGCGATCCCTGGGAAAACAGCATTCCAGGTATTAGAAGAA



TATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTT



CCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTA



ACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATG



AATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAA



TGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTT



TGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCA



CTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTAT



TGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTG



CCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAG



AAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAA



TAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGT



CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT



CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA



TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAG



CGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCT



TTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACC



GCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTC



TTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT



ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA



CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTAC



CAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG



GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG



AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCT



ACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCC



ACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG



CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAA



ACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA



CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT



ATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCT



TTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCT



GATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATAC



CGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCG



AGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCG



CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA



CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGC



TCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCT



CGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGG



AAACAGCTATGACCATGATTACGCCAAGCTCGGCGCGCCATTGG



GATGGAACCCTGCAGGCAG





Reverse
62


Complementary
AGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTA


sequence of
GAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCT


SV40polyA (SEQ
ATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAA


ID NO: 8)
CAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGG



TGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGT



ATGGCTGATTATGATC





Reverse
63


Complementary
TCAGAAGGCTGTGTTCTCAGCTTGGGCCTGGGGGCCAAAGAGCT


sequence of
GCTCAGCAACGGCCTTGCCATCATACTTGGGCAGCGTGCCCCCG


HUMAN RLBP1
AAGTCAGAGGGCAGGATGTTCTCATCGATCTCCTGGTAGAAACC


GENE CDS (SEQ
AGAAAGGTCATCCCCGTGGACAAAGACCCTCTCAAGCAGCTTGC


ID NO: 7)
TCTTCAAGAAGGGCTTGACCACATTGTAGGTCGTGGTGAAGTAC



CATGGCTGGTGGATGAAGTGGATGGCTTTGAACCGGGCTGGGAA



GGAATCCTGGAGCATGTCCACCATCTTCCTGAGATCTGAAGTCC



GGAGACTAGCAGCCTGCTGCATGGTAAAGCCCTTGAAGTTCTCA



ATGATGCAGAAGCCATTGATTTGAGTTTCCTCATTCTCCAGCAG



CTTCTCCAGGATGAAGCAATATGCCTGCAAGATCTCATCAAAGG



TGATTTCTTGACTTTGCCAGTTCTCAATGTTGAAGAGCATGACC



ACTCGGCCATACTTGTCCCGACTAGAGAGGACACCAGGGTAGCC



AGCTTCAATGGTGCAGCGGACAGCCTCTGGGGACAGGCTGTCAA



AGAGCTCAGGGTACTGCAGCCGGAAATTCACATAGCCTCTGAGC



AGCTCATAGGCACGGCCCACGTTGAACTTCCGTGCGCGGATGAA



GCGCAGGAAGAAGCCGCTGTCCTTCTCTTGCACCCTCTCCGCCA



CGGCCACCGCCAGCTCCTCCCCCGAGGCCGCCTGCGCCTGCACC



ATCTCCTGCAGCTCTCGCACTGCCTCCTCCCGGGTCTCCTCTCT



CTCGTTCAGCTCATCCTTGGCCTTCTGCAAGGTGTGGCGGGGCA



GCTGGCTGCACGGGCCAAAGACAGGTCCATGGTCCTTGGTTGTG



AGCTGCTCCAGTTGGGCACGGAGCTCCTGTTCCTCTTCAGGTAC



CATGCGGAACGTGCCCACCCCTTCTGACAT





Reverse
64


Complementary
GGTGGC


sequence of


Added KOZAK


(SEQ ID NO: 5)





Reverse
65


Complementary
GGATCCCGGGGCGGGTACAATTCCGCAGCTTTTAGAGCAGAAGT


sequence of
AACACTTCCGTACAGGCCTAGAAGTAAAGGCAACATCCACTGAG


Modified
GAGCAGTTCTTTGATTTGCACCACCACCGGATCCGGGACCTGAA


SV40INTRON (SEQ
ATAAAAGACAAAAAGACTAAACTTACCAGTTAACTTTCTGGTTT


ID NO: 4)
TTCAGTT





Reverse
66


Complementary
TCGGCAGCTCCTCCTTGGGGCTACCTGGTACCTGAATGTCCTGG


sequence of
AGCTCTAGAGGTTCCCTCCGCTGGAGGCGTGGTCCGGTCAGCAG


Human RLBP1
GTTGGGATTAGTGTGTCATAAGGAACTTCTCACCGCCCACAGTT


PROMOTER
TCCGTTAAATCGGGCTCACAGGAGGCCCTCAGTGGGGCAAAGGA


(short) (SEQ ID
AGACCCAGAGAGAAAGGGGAGAGGGGAGAGGCCTGGGCCTGGCT


NO: 3)
GGAGGCGCATCAAAGCCCTCCTTTGTGTGCTCCTGCTCTGGAGT



TCCTGCTCGGCCATGTGGAAGCCCGGCTGTGGGGCTGGGATCTG



GGCCAGTCCCATTCCCTCTTTTCTCTGCCCTCTTTCTCCTCAAG



ATCCCGGGGTGGGGTTGCTGAGAGAGCACCCCCCCCCCCCCACC



ACCACCACCAGGGTAATAAGAGGTGAAGGGAAATCGTAAATATG



ACTACATCTACAGTGGCAGCTCTGGCAAATCCAGGCCTATTGCC



CACCCCTCCCCCAGCCAGCAGGACCTGGCATGGTAGTTTTCACC



TCTGCAGTGAGTGGGGTCAGTTGAGAAATGTGGCTGGTTAAGGC



CAAGCAGGGAGAGGACAA





Reverse
67


Complementary
TTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGG


sequence of
TCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGG


eGFP (SEQ ID
TCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGG


NO: 24)
CAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGT



GGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATC



TTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGAT



ATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCA



GGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATG



CGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGT



CTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGA



CGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTC



ATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAG



GGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGG



TGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCC



TCGCCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGCC



GTCCAGCTCGACCAGGATGGGCACCACCCCGGTGAACAGCTCCT



CGCCCTTGCTCACCAT
















TABLE 2







Plasmid Composition


Plasmid Composition









SEQUENCE IDENTIFIER (SEQ. ID. NO:)AND


Elements
SEQUENCE INFORMATION










Plasmid TM017 Composition








ΔITR
 1



occurs at bp 4 through bp 106 of SEQ ID



NO: 26





Human RLBP1
 3


Promoter(short)
Occurs at bp 119 through bp 708 of SEQ ID



NO: 26





MODIFIED
 4


SV40INTRON
occurs at bp 723 through bp 905 of SEQ ID



NO: 26





Added Kozak
 5



occurs at bp 919 through bp 924 of SEQ ID



NO: 26





HUMAN RLBP1
 6


GENE CDS
occurs at bp 925 through bp 1878 of SEQ ID



NO: 26





SV40 POLYA
 8



occurs at bp 1937 through bp 2172 of SEQ



ID NO: 26





3′ ITR
 9



occurs at bp 2201 through bp 2330 of SEQ



ID NO: 26





AMP BACTERIAL
15


BACKBONE
occurs at bp 2331 through bp 4949 of SEQ



ID NO: 26





TM017 PLASMID
26


SEQUENCE
ctgcgcgctcgctcgctcactgaggccgcccgggcaaagccc



gggcgtcgggcgacctttggtcgcccggcctcagtgagcgag



cgagcgcgcagagagggagtggggtaccacgcgtttgtcctc



tccctgcttggccttaaccagccacatttctcaactgacccc



actcactgcagaggtgaaaactaccatgccaggtcctgctgg



ctgggggaggggtgggcaataggcctggatttgccagagctg



ccactgtagatgtagtcatatttacgatttcccttcacctct



tattaccctggtggtggtggtgggggggggggggtgctctct



cagcaaccccaccccgggatcttgaggagaaagagggcagag



aaaagagggaatgggactggcccagatcccagccccacagcc



gggcttccacatggccgagcaggaactccagagcaggagcac



acaaaggagggctttgatgcgcctccagccaggcccaggcct



ctcccctctcccctttctctctgggtcttcctttgccccact



gagggcctcctgtgagcccgatttaacggaaactgtgggcgg



tgagaagttccttatgacacactaatcccaacctgctgaccg



gaccacgcctccagcggagggaacctctagagctccaggaca



ttcaggtaccaggtagccccaaggaggagctgccgaatcgat



ggatcgggaactgaaaaaccagaaagttaactggtaagttta



gtctttttgtcttttatttcaggtcccggatccggtggtggt



gcaaatcaaagaactgctcctcagtggatgttgcctttactt



ctaggcctgtacggaagtgttacttctgctctaaaagctgcg



gaattgtacccgccccgggatccatcgattgaattcgccacc



atgtcagaaggggtgggcacgttccgcatggtacctgaagag



gaacaggagctccgtgcccaactggagcagctcacaaccaag



gaccatggacctgtctttggcccgtgcagccagctgccccgc



cacaccttgcagaaggccaaggatgagctgaacgagagagag



gagacccgggaggaggcagtgcgagagctgcaggagatggtg



caggcgcaggcggcctcgggggaggagctggcggtggccgtg



gcggagagggtgcaagagaaggacagcggcttcttcctgcgc



ttcatccgcgcacggaagttcaacgtgggccgtgcctatgag



ctgctcagaggctatgtgaatttccggctgcagtaccctgag



ctctttgacagcctgtccccagaggctgtccgctgcaccatt



gaagctggctaccctggtgtcctctctagtcgggacaagtat



ggccgagtggtcatgctcttcaacattgagaactggcaaagt



caagaaatcacctttgatgagatcttgcaggcatattgcttc



atcctggagaagctgctggagaatgaggaaactcaaatcaat



ggcttctgcatcattgagaacttcaagggctttaccatgcag



caggctgctagtctccggacttcagatctcaggaagatggtg



gacatgctccaggattccttcccagcccggttcaaagccatc



cacttcatccaccagccatggtacttcaccacgacctacaat



gtggtcaagcccttcttgaagagcaagctgcttgagagggtc



tttgtccacggggatgacctttctggtttctaccaggagatc



gatgagaacatcctgccctctgacttcgggggcacgctgccc



aagtatgatggcaaggccgttgctgagcagctctttggcccc



caggcccaagctgagaacacagccttctgaggatcgtaccgg



tcgacctgcagaagcttgcctcgagcagcgctgctcgagaga



tctggatcataatcagccataccacatttgtagaggttttac



ttgctttaaaaaacctcccacacctccccctgaacctgaaac



ataaaatgaatgcaattgttgttgttaacttgtttattgcag



cttataatggttacaaataaagcaatagcatcacaaatttca



caaataaagcatttttttcactgcattctagttgtggtttgt



ccaaactcatcaatgtatcttatcatgtctggtaaccacgtg



cggaccgagcggccgcaggaacccctagtgatggagttggcc



actccctctctgcgcgctcgctcgctcactgaggccgggcga



ccaaaggtcgcccgacgcccgggctttgcccgggcggcctca



gtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatg



cggtattttctccttacgcatctgtgcggtatttcacaccgc



atacgtcaaagcaaccatagtacgcgccctgtagcggcgcat



taagcgcggcgggtgtggtggttacgcgcagcgtgaccgcta



cacttgccagcgccttagcgcccgctcctttcgctttcttcc



cttcctttctcgccacgttcgccggctttccccgtcaagctc



taaatcgggggctccctttagggttccgatttagtgctttac



ggcacctcgaccccaaaaaacttgatttgggtgatggttcac



gtagtgggccatcgccctgatagacggtttttcgccctttga



cgttggagtccacgttctttaatagtggactcttgttccaaa



ctggaacaacactcaactctatctcgggctattcttttgatt



tataagggattttgccgatttcggtctattggttaaaaaatg



agctgatttaacaaaaatttaacgcgaattttaacaaaatat



taacgtttacaattttatggtgcactctcagtacaatctgct



ctgatgccgcatagttaagccagccccgacacccgccaacac



ccgctgacgcgccctgacgggcttgtctgctcccggcatccg



cttacagacaagctgtgaccgtctccgggagctgcatgtgtc



agaggttttcaccgtcatcaccgaaacgcgcgagacgaaagg



gcctcgtgatacgcctatttttataggttaatgtcatgataa



taatggtttcttagacgtcaggtggcacttttcggggaaatg



tgcgcggaacccctatttgtttatttttctaaatacattcaa



atatgtatccgctcatgagacaataaccctgataaatgcttc



aataatattgaaaaaggaagagtatgagtattcaacatttcc



gtgtcgcccttattcccttttttgcggcattttgccttcctg



tttttgctcacccagaaacgctggtgaaagtaaaagatgctg



aagatcagttgggtgcacgagtgggttacatcgaactggatc



tcaacagcggtaagatccttgagagttttcgccccgaagaac



gttttccaatgatgagcacttttaaagttctgctatgtggcg



cggtattatcccgtattgacgccgggcaagagcaactcggtc



gccgcatacactattctcagaatgacttggttgagtactcac



cagtcacagaaaagcatcttacggatggcatgacagtaagag



aattatgcagtgctgccataaccatgagtgataacactgcgg



ccaacttacttctgacaacgatcggaggaccgaaggagctaa



ccgcttttttgcacaacatgggggatcatgtaactcgccttg



atcgttgggaaccggagctgaatgaagccataccaaacgacg



agcgtgacaccacgatgcctgtagcaatggcaacaacgttgc



gcaaactattaactggcgaactacttactctagcttcccggc



aacaattaatagactggatggaggcggataaagttgcaggac



cacttctgcgctcggcccttccggctggctggtttattgctg



ataaatctggagccggtgagcgtgggtctcgcggtatcattg



cagcactggggccagatggtaagccctcccgtatcgtagtta



tctacacgacggggagtcaggcaactatggatgaacgaaata



gacagatcgctgagataggtgcctcactgattaagcattggt



aactgtcagaccaagtttactcatatatactttagattgatt



taaaacttcatttttaatttaaaaggatctaggtgaagatcc



tttttgataatctcatgaccaaaatcccttaacgtgagtttt



cgttccactgagcgtcagaccccgtagaaaagatcaaaggat



cttcttgaaatcctttttttctgcgcgtaatctgctgcttgc



aaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg



atcaagagctaccaactctttttccgaaggtaactggcttca



gcagagcgcagataccaaatactgttcttctagtgtagccgt



agttaggccaccacttcaagaactctgtagcaccgcctacat



acctcgctctgctaatcctgttaccagtggctgctgccagtg



gcgataagtcgtgtcttaccgggttggactcaagacgatagt



taccggataaggcgcagcggtcgggctgaacggggggttcgt



gcacacagcccagcttggagcgaacgacctacaccgaactga



gatacctacagcgtgagctatgagaaagcgccacgcttcccg



aagggagaaaggcggacaggtatccggtaagcggcagggtcg



gaacaggagagcgcacgagggagcttccagggggaaacgcct



ggtatctttatagtcctgtcgggtttcgccacctctgacttg



agcgtcgatttttgtgatgctcgtcaggggggcggagcctat



ggaaaaacgccagcaacgcggcctttttacggttcctggcct



tttgctggccttttgctcacatgtcctgcaggcag





GENE CASSETTE
51


OF PLASMID
cgcgctcgctcgctcactgaggccgcccgggcaaagcccggg


TM017 OCCURS AT
cgtcgggcgacctttggtcgcccggcctcagtgagcgagcga


BP 4 THROUGH
gcgcgcagagagggagtggggtaccacgcgtttgtcctctcc


2330 OF SEQ ID
ctgcttggccttaaccagccacatttctcaactgaccccact


NO: 26
cactgcagaggtgaaaactaccatgccaggtcctgctggctg



ggggaggggtgggcaataggcctggatttgccagagctgcca



ctgtagatgtagtcatatttacgatttcccttcacctcttat



taccctggtggtggtggtgggggggggggggtgctctctcag



caaccccaccccgggatcttgaggagaaagagggcagagaaa



agagggaatgggactggcccagatcccagccccacagccggg



cttccacatggccgagcaggaactccagagcaggagcacaca



aaggagggctttgatgcgcctccagccaggcccaggcctctc



ccctctcccctttctctctgggtcttcctttgccccactgag



ggcctcctgtgagcccgatttaacggaaactgtgggcggtga



gaagttccttatgacacactaatcccaacctgctgaccggac



cacgcctccagcggagggaacctctagagctccaggacattc



aggtaccaggtagccccaaggaggagctgccgaatcgatgga



tcgggaactgaaaaaccagaaagttaactggtaagtttagtc



tttttgtcttttatttcaggtcccggatccggtggtggtgca



aatcaaagaactgctcctcagtggatgttgcctttacttcta



ggcctgtacggaagtgttacttctgctctaaaagctgcggaa



ttgtacccgccccgggatccatcgattgaattcgccaccatg



tcagaaggggtgggcacgttccgcatggtacctgaagaggaa



caggagctccgtgcccaactggagcagctcacaaccaaggac



catggacctgtctttggcccgtgcagccagctgccccgccac



accttgcagaaggccaaggatgagctgaacgagagagaggag



acccgggaggaggcagtgcgagagctgcaggagatggtgcag



gcgcaggcggcctcgggggaggagctggcggtggccgtggcg



gagagggtgcaagagaaggacagcggcttcttcctgcgcttc



atccgcgcacggaagttcaacgtgggccgtgcctatgagctg



ctcagaggctatgtgaatttccggctgcagtaccctgagctc



tttgacagcctgtccccagaggctgtccgctgcaccattgaa



gctggctaccctggtgtcctctctagtcgggacaagtatggc



cgagtggtcatgctcttcaacattgagaactggcaaagtcaa



gaaatcacctttgatgagatcttgcaggcatattgcttcatc



ctggagaagctgctggagaatgaggaaactcaaatcaatggc



ttctgcatcattgagaacttcaagggctttaccatgcagcag



gctgctagtctccggacttcagatctcaggaagatggtggac



atgctccaggattccttcccagcccggttcaaagccatccac



ttcatccaccagccatggtacttcaccacgacctacaatgtg



gtcaagcccttcttgaagagcaagctgcttgagagggtcttt



gtccacggggatgacctttctggtttctaccaggagatcgat



gagaacatcctgccctctgacttcgggggcacgctgcccaag



tatgatggcaaggccgttgctgagcagctctttggcccccag



gcccaagctgagaacacagccttctgaggatcgtaccggtcg



acctgcagaagcttgcctcgagcagcgctgctcgagagatct



ggatcataatcagccataccacatttgtagaggttttacttg



ctttaaaaaacctcccacacctccccctgaacctgaaacata



aaatgaatgcaattgttgttgttaacttgtttattgcagctt



ataatggttacaaataaagcaatagcatcacaaatttcacaa



ataaagcatttttttcactgcattctagttgtggtttgtcca



aactcatcaatgtatcttatcatgtctggtaaccacgtgcgg



accgagcggccgcaggaacccctagtgatggagttggccact



ccctctctgcgcgctcgctcgctcactgaggccgggcgacca



aaggtcgcccgacgcccgggctttgcccgggcggcctcagtg



agcgagcgagcgcgcag










Plasmid TM037 Composition








5′ ITR
 2



occurs @ bp 1 through bp 119 of SEQ ID NO:



27





Human RLBP1
10


Promoter(long)
occurs @ bp 137 through bp 3293 of SEQ ID



NO: 27





Added Kozak
 5



occurs at bp 3300 through bp 3305 of SEQ



ID NO: 27





HUMAN RLBP1
 6


GENE CDS
occurs at bp 3306 through bp 4259 of SEQ



ID NO: 27





SV40 POLYA
 8



occurs at bp 4318 through bp 4553 of SEQ



ID NO: 27





3′ ITR
 9



occurs at bp 4582 through bp 4711 of SEQ



ID NO: 27





AMP BACTERIAL
15


BACKBONE
occurs at bp 4712 through bp 7330 of SEQ



ID NO: 27





Plasmid TM037
27


sequence
CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGC



GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAG



AGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCG



CACGCAGCTTTTGTCCTCTCCCTGCTTGGCCTTAACCAGCCA



CATTTCTCAACTGACCCCACTCACTGCAGAGGTGAAAACTAC



CATGCCAGGTCCTGCTGGCTGGGGGAGGGGTGGGCAATAGGC



CTGGATTTGCCAGAGCTGCCACTGTAGATGTAGTCATATTTA



CGATTTCCCTTCACCTCTTATTACCCTGGTGGTGGTGGTGGG



GGGGGGGGGGTGCTCTCTCAGCAACCCCACCCCGGGATCTTG



AGGAGAAAGAGGGCAGAGAAAAGAGGGAATGGGACTGGCCCA



GATCCCAGCCCCACAGCCGGGCTTCCACATGGCCGAGCAGGA



ACTCCAGAGCAGGAGCACACAAAGGAGGGCTTTGATGCGCCT



CCAGCCAGGCCCAGGCCTCTCCCCTCTCCCCTTTCTCTCTGG



GTCTTCCTTTGCCCCACTGAGGGCCTCCTGTGAGCCCGATTT



AACGGAAACTGTGGGCGGTGAGAAGTTCCTTATGACACACTA



ATCCCAACCTGCTGACCGGACCACGCCTCCAGCGGAGGGAAC



CTCTAGAGCTCCAGGACATTCAGGTACCAGGTAGCCCCAAGG



AGGAGCTGCCGACCTGGCAGGTAAGTCAATACCTGGGGCTTG



CCTGGGCCAGGGAGCCCAGGACTGGGGTGAGGACTCAGGGGA



GCAGGGAGACCACGTCCCAAGATGCCTGTAAAACTGAAACCA



CCTGGCCATTCTCCAGGTTGAGCCAGACCAATTTGATGGCAG



ATTTAGCAAATAAAAATACAGGACACCCAGTTAAATGTGAAT



TTCAGATGAACAGCAAATACTTTTTTAGTATTAAAAAAGTTC



ACATTTAGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCC



GAGGCAGGCAGATCACCTGAGGTCAGGAGTTCGAGACCAGCC



TGGCCAACATGGTGAAACCCCATCTCCACTAAAAATACCAAA



AATTAGCCAGGCGTGCTGGTGGGCACCTGTAGTTCCAGCTAC



TCAGGAGGCTAAGGCAGGAGAATTGCTTGAACCTGGGAGGCA



GAGGTTGCAGTGAGCTGAGATCGCACCATTGCACTCTAGCCT



GGGCGACAAGAACAAAACTCCATCTCAAAAAAAAAAAAAAAA



AAAAAGTTCACATTTAACTGGGCATTCTGTATTTAATTGGTA



ATCTGAGATGGCAGGGAACAGCATCAGCATGGTGTGAGGGAT



AGGCATTTTTTCATTGTGTACAGCTTGTAAATCAGTATTTTT



AAAACTCAAAGTTAATGGCTTGGGCATATTTAGAAAAGAGTT



GCCGCACGGACTTGAACCCTGTATTCCTAAAATCTAGGATCT



TGTTCTGATGGTCTGCACAACTGGCTGGGGGTGTCCAGCCAC



TGTCCCTCTTGCCTGGGCTCCCCAGGGCAGTTCTGTCAGCCT



CTCCATTTCCATTCCTGTTCCAGCAAAACCCAACTGATAGCA



CAGCAGCATTTCAGCCTGTCTACCTCTGTGCCCACATACCTG



GATGTCTACCAGCCAGAAAGGTGGCTTAGATTTGGTTCCTGT



GGGTGGATTATGGCCCCCAGAACTTCCCTGTGCTTGCTGGGG



GTGTGGAGTGGAAAGAGCAGGAAATGGGGGACCCTCCGATAC



TCTATGGGGGTCCTCCAAGTCTCTTTGTGCAAGTTAGGGTAA



TAATCAATATGGAGCTAAGAAAGAGAAGGGGAACTATGCTTT



AGAACAGGACACTGTGCCAGGAGCATTGCAGAAATTATATGG



TTTTCACGACAGTTCTTTTTGGTAGGTACTGTTATTATCCTC



AGTTTGCAGATGAGGAAACTGAGACCCAGAAAGGTTAAATAA



CTTGCTAGGGTCACACAAGTCATAACTGACAAAGCCTGATTC



AAACCCAGGTCTCCCTAACCTTTAAGGTTTCTATGACGCCAG



CTCTCCTAGGGAGTTTGTCTTCAGATGTCTTGGCTCTAGGTG



TCAAAAAAAGACTTGGTGTCAGGCAGGCATAGGTTCAAGTCC



CAACTCTGTCACTTACCAACTGTGACTAGGTGATTGAACTGA



CCATGGAACCTGGTCACATGCAGGAGCAGGATGGTGAAGGGT



TCTTGAAGGCACTTAGGCAGGACATTTAGGCAGGAGAGAAAA



CCTGGAAACAGAAGAGCTGTCTCCAAAAATACCCACTGGGGA



AGCAGGTTGTCATGTGGGCCATGAATGGGACCTGTTCTGGTA



ACCAAGCATTGCTTATGTGTCCATTACATTTCATAACACTTC



CATCCTACTTTACAGGGAACAACCAAGACTGGGGTTAAATCT



CACAGCCTGCAAGTGGAAGAGAAGAACTTGAACCCAGGTCCA



ACTTTTGCGCCACAGCAGGCTGCCTCTTGGTCCTGACAGGAA



GTCACAACTTGGGTCTGAGTACTGATCCCTGGCTATTTTTTG



GCTGTGTTACCTTGGACAAGTCACTTATTCCTCCTCCCGTTT



CCTCCTATGTAAAATGGAAATAATAATGTTGACCCTGGGTCT



GAGAGAGTGGATTTGAAAGTACTTAGTGCATCACAAAGCACA



GAACACACTTCCAGTCTCGTGATTATGTACTTATGTAACTGG



TCATCACCCATCTTGAGAATGAATGCATTGGGGAAAGGGCCA



TCCACTAGGCTGCGAAGTTTCTGAGGGACTCCTTCGGGCTGG



AGAAGGATGGCCACAGGAGGGAGGAGAGATTGCCTTATCCTG



CAGTGATCATGTCATTGAGAACAGAGCCAGATTCTTTTTTTC



CTGGCAGGGCCAACTTGTTTTAACATCTAAGGACTGAGCTAT



TTGTGTCTGTGCCCTTTGTCCAAGCAGTGTTTCCCAAAGTGT



AGCCCAAGAACCATCTCCCTCAGAGCCACCAGGAAGTGCTTT



AAATTGCAGGTTCCTAGGCCACAGCCTGCACCTGCAGAGTCA



GAATCATGGAGGTTGGGACCCAGGCACCTGCGTTTCTAACAA



ATGCCTCGGGTGATTCTGATGCAATTGAAAGTTTGAGATCCA



CAGTTCTGAGACAATAACAGAATGGTTTTTCTAACCCCTGCA



GCCCTGACTTCCTATCCTAGGGAAGGGGCCGGCTGGAGAGGC



CAGGACAGAGAAAGCAGATCCCTTCTTTTTCCAAGGACTCTG



TGTCTTCCATAGGCAACGAATTCGCCACCATGTCAGAAGGGG



TGGGCACGTTCCGCATGGTACCTGAAGAGGAACAGGAGCTCC



GTGCCCAACTGGAGCAGCTCACAACCAAGGACCATGGACCTG



TCTTTGGCCCGTGCAGCCAGCTGCCCCGCCACACCTTGCAGA



AGGCCAAGGATGAGCTGAACGAGAGAGAGGAGACCCGGGAGG



AGGCAGTGCGAGAGCTGCAGGAGATGGTGCAGGCGCAGGCGG



CCTCGGGGGAGGAGCTGGCGGTGGCCGTGGCGGAGAGGGTGC



AAGAGAAGGACAGCGGCTTCTTCCTGCGCTTCATCCGCGCAC



GGAAGTTCAACGTGGGCCGTGCCTATGAGCTGCTCAGAGGCT



ATGTGAATTTCCGGCTGCAGTACCCTGAGCTCTTTGACAGCC



TGTCCCCAGAGGCTGTCCGCTGCACCATTGAAGCTGGCTACC



CTGGTGTCCTCTCTAGTCGGGACAAGTATGGCCGAGTGGTCA



TGCTCTTCAACATTGAGAACTGGCAAAGTCAAGAAATCACCT



TTGATGAGATCTTGCAGGCATATTGCTTCATCCTGGAGAAGC



TGCTGGAGAATGAGGAAACTCAAATCAATGGCTTCTGCATCA



TTGAGAACTTCAAGGGCTTTACCATGCAGCAGGCTGCTAGTC



TCCGGACTTCAGATCTCAGGAAGATGGTGGACATGCTCCAGG



ATTCCTTCCCAGCCCGGTTCAAAGCCATCCACTTCATCCACC



AGCCATGGTACTTCACCACGACCTACAATGTGGTCAAGCCCT



TCTTGAAGAGCAAGCTGCTTGAGAGGGTCTTTGTCCACGGGG



ATGACCTTTCTGGTTTCTACCAGGAGATCGATGAGAACATCC



TGCCCTCTGACTTCGGGGGCACGCTGCCCAAGTATGATGGCA



AGGCCGTTGCTGAGCAGCTCTTTGGCCCCCAGGCCCAAGCTG



AGAACACAGCCTTCTGAGGATCGTACCGGTCGACCTGCAGAA



GCTTGCCTCGAGCAGCGCTGCTCGAGAGATCTGGATCATAAT



CAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAA



CCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGC



AATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTA



CAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATT



TTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAA



TGTATCTTATCATGTCTGGTAACCACGTGCGGACCGAGCGGC



CGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGC



GCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC



GACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG



CGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCC



TTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCA



ACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGG



TGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGC



CTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGC



CACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCT



CCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCC



CAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATC



GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCAC



GTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT



CAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTT



GCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACA



AAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAAT



TTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA



GTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCC



CTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGC



TGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACC



GTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACG



CCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTA



GACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCC



TATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT



CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAA



AAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT



TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCC



AGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGG



TGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAA



GATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT



GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCG



TATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTA



TTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA



GCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC



TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT



GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA



CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACC



GGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCAC



GATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAAC



TGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGA



CTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTC



GGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC



CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCC



AGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG



GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA



GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA



AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTT



TTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT



CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGC



GTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAAATCC



TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC



ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC



AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT



ACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCA



CTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT



AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG



TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC



GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAG



CTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG



TGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGC



GGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG



CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG



TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT



GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAG



CAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTT



TGCTCACATGTCCTGCAGGCAG





GENE CASSETTE
52


OF PLASMID
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


TM037 OCCURS AT
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


BP 1 THROUGH
agagggagtggccaactccatcactaggggttcctgcggccg


4711 OF SEQ ID
cacgcagcttttgtcctctccctgcttggccttaaccagcca


NO: 27
catttctcaactgaccccactcactgcagaggtgaaaactac



catgccaggtcctgctggctgggggaggggtgggcaataggc



ctggatttgccagagctgccactgtagatgtagtcatattta



cgatttcccttcacctcttattaccctggtggtggtggtggg



ggggggggggtgctctctcagcaaccccaccccgggatcttg



aggagaaagagggcagagaaaagagggaatgggactggccca



gatcccagccccacagccgggcttccacatggccgagcagga



actccagagcaggagcacacaaaggagggctttgatgcgcct



ccagccaggcccaggcctctcccctctcccctttctctctgg



gtcttcctttgccccactgagggcctcctgtgagcccgattt



aacggaaactgtgggcggtgagaagttccttatgacacacta



atcccaacctgctgaccggaccacgcctccagcggagggaac



ctctagagctccaggacattcaggtaccaggtagccccaagg



aggagctgccgacctggcaggtaagtcaatacctggggcttg



cctgggccagggagcccaggactggggtgaggactcagggga



gcagggagaccacgtcccaagatgcctgtaaaactgaaacca



cctggccattctccaggttgagccagaccaatttgatggcag



atttagcaaataaaaatacaggacacccagttaaatgtgaat



ttcagatgaacagcaaatacttttttagtattaaaaaagttc



acatttaggctcacgcctgtaatcccagcactttgggaggcc



gaggcaggcagatcacctgaggtcaggagttcgagaccagcc



tggccaacatggtgaaaccccatctccactaaaaataccaaa



aattagccaggcgtgctggtgggcacctgtagttccagctac



tcaggaggctaaggcaggagaattgcttgaacctgggaggca



gaggttgcagtgagctgagatcgcaccattgcactctagcct



gggcgacaagaacaaaactccatctcaaaaaaaaaaaaaaaa



aaaaagttcacatttaactgggcattctgtatttaattggta



atctgagatggcagggaacagcatcagcatggtgtgagggat



aggcattttttcattgtgtacagcttgtaaatcagtattttt



aaaactcaaagttaatggcttgggcatatttagaaaagagtt



gccgcacggacttgaaccctgtattcctaaaatctaggatct



tgttctgatggtctgcacaactggctgggggtgtccagccac



tgtccctcttgcctgggctccccagggcagttctgtcagcct



ctccatttccattcctgttccagcaaaacccaactgatagca



cagcagcatttcagcctgtctacctctgtgcccacatacctg



gatgtctaccagccagaaaggtggcttagatttggttcctgt



gggtggattatggcccccagaacttccctgtgcttgctgggg



gtgtggagtggaaagagcaggaaatgggggaccctccgatac



tctatgggggtcctccaagtctctttgtgcaagttagggtaa



taatcaatatggagctaagaaagagaaggggaactatgcttt



agaacaggacactgtgccaggagcattgcagaaattatatgg



ttttcacgacagttctttttggtaggtactgttattatcctc



agtttgcagatgaggaaactgagacccagaaaggttaaataa



cttgctagggtcacacaagtcataactgacaaagcctgattc



aaacccaggtctccctaacctttaaggtttctatgacgccag



ctctcctagggagtttgtcttcagatgtcttggctctaggtg



tcaaaaaaagacttggtgtcaggcaggcataggttcaagtcc



caactctgtcacttaccaactgtgactaggtgattgaactga



ccatggaacctggtcacatgcaggagcaggatggtgaagggt



tcttgaaggcacttaggcaggacatttaggcaggagagaaaa



cctggaaacagaagagctgtctccaaaaatacccactgggga



agcaggttgtcatgtgggccatgaatgggacctgttctggta



accaagcattgcttatgtgtccattacatttcataacacttc



catcctactttacagggaacaaccaagactggggttaaatct



cacagcctgcaagtggaagagaagaacttgaacccaggtcca



acttttgcgccacagcaggctgcctcttggtcctgacaggaa



gtcacaacttgggtctgagtactgatccctggctattttttg



gctgtgttaccttggacaagtcacttattcctcctcccgttt



cctcctatgtaaaatggaaataataatgttgaccctgggtct



gagagagtggatttgaaagtacttagtgcatcacaaagcaca



gaacacacttccagtctcgtgattatgtacttatgtaactgg



tcatcacccatcttgagaatgaatgcattggggaaagggcca



tccactaggctgcgaagtttctgagggactccttcgggctgg



agaaggatggccacaggagggaggagagattgccttatcctg



cagtgatcatgtcattgagaacagagccagattctttttttc



ctggcagggccaacttgttttaacatctaaggactgagctat



ttgtgtctgtgccctttgtccaagcagtgtttcccaaagtgt



agcccaagaaccatctccctcagagccaccaggaagtgcttt



aaattgcaggttcctaggccacagcctgcacctgcagagtca



gaatcatggaggttgggacccaggcacctgcgtttctaacaa



atgcctcgggtgattctgatgcaattgaaagtttgagatcca



cagttctgagacaataacagaatggtttttctaacccctgca



gccctgacttcctatcctagggaaggggccggctggagaggc



caggacagagaaagcagatcccttctttttccaaggactctg



tgtcttccataggcaacgaattcgccaccatgtcagaagggg



tgggcacgttccgcatggtacctgaagaggaacaggagctcc



gtgcccaactggagcagctcacaaccaaggaccatggacctg



tctttggcccgtgcagccagctgccccgccacaccttgcaga



aggccaaggatgagctgaacgagagagaggagacccgggagg



aggcagtgcgagagctgcaggagatggtgcaggcgcaggcgg



cctcgggggaggagctggcggtggccgtggcggagagggtgc



aagagaaggacagcggcttcttcctgcgcttcatccgcgcac



ggaagttcaacgtgggccgtgcctatgagctgctcagaggct



atgtgaatttccggctgcagtaccctgagctctttgacagcc



tgtccccagaggctgtccgctgcaccattgaagctggctacc



ctggtgtcctctctagtcgggacaagtatggccgagtggtca



tgctcttcaacattgagaactggcaaagtcaagaaatcacct



ttgatgagatcttgcaggcatattgcttcatcctggagaagc



tgctggagaatgaggaaactcaaatcaatggcttctgcatca



ttgagaacttcaagggctttaccatgcagcaggctgctagtc



tccggacttcagatctcaggaagatggtggacatgctccagg



attccttcccagcccggttcaaagccatccacttcatccacc



agccatggtacttcaccacgacctacaatgtggtcaagccct



tcttgaagagcaagctgcttgagagggtctttgtccacgggg



atgacctttctggtttctaccaggagatcgatgagaacatcc



tgccctctgacttcgggggcacgctgcccaagtatgatggca



aggccgttgctgagcagctctttggcccccaggcccaagctg



agaacacagccttctgaggatcgtaccggtcgacctgcagaa



gcttgcctcgagcagcgctgctcgagagatctggatcataat



cagccataccacatttgtagaggttttacttgctttaaaaaa



cctcccacacctccccctgaacctgaaacataaaatgaatgc



aattgttgttgttaacttgtttattgcagcttataatggtta



caaataaagcaatagcatcacaaatttcacaaataaagcatt



tttttcactgcattctagttgtggtttgtccaaactcatcaa



tgtatcttatcatgtctggtaaccacgtgcggaccgagcggc



cgcaggaacccctagtgatggagttggccactccctctctgc



gcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc



gacgcccgggctttgcccgggcggcctcagtgagcgagcgag



cgcgcag










Plasmid AG007 Composition








5′ ITR
 2



occurs @ bp 1 through bp 119 of SEQ ID



NO: 28





Human RPE65
11


Promoter
occurs @ bp 134 through bp 1718 of SEQ ID



NO: 28





ADDED-KOZAK
 5



occurs @ bp 1725 through bp 1730 of SEQ ID



NO: 28





HUMAN RLBP1
 6


GENE CDS
occurs at bp 1731 through bp 2684 of SEQ



ID NO: 28





SV40 POLYA
 8



occurs at bp 2742 through bp 2977 of SEQ



ID NO: 28





RLBP1 INTRONIC
14


SEQUENCE AS
occurs at bp 2985 through bp 4487 of SEQ


STUFFER
ID NO: 28


SEQUENCE





3′ ITR
 9



occurs at bp 4516 through bp 4645 of SEQ



ID NO: 28





AMP BACTERIAL
15


BACKBONE
occurs at bp 4646 through bp 7264 of SEQ



ID NO: 28





AG007 Plasmid
28


Sequence
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc



gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag



agagggagtggccaactccatcactaggggttcctgcggccg



cacgcgttacgtaatatttattgaagtttaatattgtgtttg



tgatacagaagtatttgctttaattctaaataaaaattttat



gcttttattgctggtttaagaagatttggattatccttgtac



tttgaggagaagtttcttatttgaaatattttggaaacaggt



cttttaatgtggaaagatagatattaatctcctcttctatta



ctctccaagatccaacaaaagtgattataccccccaaaatat



gatggtagtatcttatactaccatcattttataggcataggg



ctcttagctgcaaataatggaactaactctaataaagcagaa



cgcaaatattgtaaatattagagagctaacaatctctgggat



ggctaaaggatggagcttggaggctacccagccagtaacaat



attccgggctccactgttgaatggagacactacaactgcctt



ggatgggcagagatattatggatgctaagccccaggtgctac



cattaggacttctaccactgtccctaacgggtggagcccatc



acatgcctatgccctcactgtaaggaaatgaagctactgttg



tatatcttgggaagcacttggattaattgttatacagttttg



ttgaagaagacccctagggtaagtagccataactgcacacta



aatttaaaattgttaatgagtttctcaaaaaaaatgttaagg



ttgttagctggtatagtatatatcttgcctgttttccaagga



cttctttgggcagtaccttgtctgtgctggcaagcaactgag



acttaatgaaagagtattggagatatgaatgaattgatgctg



tatactctcagagtgccaaacatataccaatggacaagaagg



tgaggcagagagcagacaggcattagtgacaagcaaagatat



gcagaatttcattctcagcaaatcaaaagtcctcaacctggt



tggaagaatattggcactgaatggtatcaataaggttgctag



agagggttagaggtgcacaatgtgcttccataacattttata



cttctccaatcttagcactaatcaaacatggttgaatacttt



gtttactataactcttacagagttataagatctgtgaagaca



gggacagggacaatacccatctctgtctggttcataggtggt



atgtaatagatatttttaaaaataagtgagttaatgaatgag



ggtgagaatgaaggcacagaggtattagggggaggtgggccc



cagagaatggtgccaaggtccagtggggtgactgggatcagc



tcaggcctgacgctggccactcccacctagctcctttctttc



taatctgttctcattctccttgggaaggattgaggtctctgg



aaaacagccaaacaactgttatgggaacagcaagcccaaata



aagccaagcatcagggggatctgagagctgaaagcaacttct



gttccccctccctcagctgaaggggtggggaagggctcccaa



agccataactccttttaagggatttagaaggcataaaaaggc



ccctggctgagaacttccttcttcattctgcagttggtgaat



tcgccaccatgtcagaaggggtgggcacgttccgcatggtac



ctgaagaggaacaggagctccgtgcccaactggagcagctca



caaccaaggaccatggacctgtctttggcccgtgcagccagc



tgccccgccacaccttgcagaaggccaaggatgagctgaacg



agagagaggagacccgggaggaggcagtgcgagagctgcagg



agatggtgcaggcgcaggcggcctcgggggaggagctggcgg



tggccgtggcggagagggtgcaagagaaggacagcggcttct



tcctgcgcttcatccgcgcacggaagttcaacgtgggccgtg



cctatgagctgctcagaggctatgtgaatttccggctgcagt



accctgagctctttgacagcctgtccccagaggctgtccgct



gcaccattgaagctggctaccctggtgtcctctctagtcggg



acaagtatggccgagtggtcatgctcttcaacattgagaact



ggcaaagtcaagaaatcacctttgatgagatcttgcaggcat



attgcttcatcctggagaagctgctggagaatgaggaaactc



aaatcaatggcttctgcatcattgagaacttcaagggcttta



ccatgcagcaggctgctagtctccggacttcagatctcagga



agatggtggacatgctccaggattccttcccagcccggttca



aagccatccacttcatccaccagccatggtacttcaccacga



cctacaatgtggtcaagcccttcttgaagagcaagctgcttg



agagggtctttgtccacggggatgacctttctggtttctacc



aggagatcgatgagaacatcctgccctctgacttcgggggca



cgctgcccaagtatgatggcaaggccgttgctgagcagctct



ttggcccccaggcccaagctgagaacacagccttctgaggat



ctaccggtcgacctgcagaagcttgcctcgagcagcgctgct



cgagagatctggatcataatcagccataccacatttgtagag



gttttacttgctttaaaaaacctcccacacctccccctgaac



ctgaaacataaaatgaatgcaattgttgttgttaacttgttt



attgcagcttataatggttacaaataaagcaatagcatcaca



aatttcacaaataaagcatttttttcactgcattctagttgt



ggtttgtccaaactcatcaatgtatcttatcatgtctggtaa



ccattctccaggttgagccagaccaatttgatggtagattta



gcaaataaaaatacaggacacccagttaaatgtgaatttccg



atgaacagcaaatacttttttagtattaaaaaagttcacatt



taggctcacgcctgtaatcccagcactttgggaggccgaggc



aggcagatcacctgaggtcaggagttcgagaccagcctggcc



aacatggtgaaaccccatctccactaaaaataccaaaaatta



gccaggcgtgctggtgggcacctgtagttccagctactcagg



aggctaaggcaggagaattgcttgaacctgggaggcagaggt



tgcagtgagctgagatcgcaccattgcactctagcctgggcg



acaagaacaaaactccatctcaaaaaaaaaaaaaaaaaaaaa



gttcacatttaactgggcattctgtatttaattggtaatctg



agatggcagggaacagcatcagcatggtgtgagggataggca



ttttttcattgtgtacagcttgtaaatcagtatttttaaaac



tcaaagttaatggcttgggcatatttagaaaagagttgccgc



acggacttgaaccctgtattcctaaaatctaggatcttgttc



tgatggtctgcacaactggctgggggtgtccagccactgtcc



ctcttgcctgggctccccagggcagttctgtcagcctctcca



tttccattcctgttccagcaaaacccaactgatagcacagca



gcatttcagcctgtctacctctgtgcccacatacctggatgt



ctaccagccagaaaggtggcttagatttggttcctgtgggtg



gattatggcccccagaacttccctgtgcttgctgggggtgtg



gagtggaaagagcaggaaatgggggaccctccgatactctat



gggggtcctccaagtctctttgtgcaagttagggtaataatc



aatatggagctaagaaagagaaggggaactatgctttagaac



aggacactgtgccaggagcattgcagaaattatatggttttc



acgacagttctttttggtaggtactgttattatcctcagttt



gcagatgaggaaactgagacccagaaaggttaaataacttgc



tagggtcacacaagtcataactgacaaagcctgattcaaacc



caggtctccctaacctttaaggtttctatgacgccagctctc



ctagggagtttgtcttcagatgtcttggctctaggtgtcaaa



aaaagacttggtgtcaggcaggcataggttcaagtcccaact



ctgtcacttaccaactgtgactaggtgattgaactgaccatg



gaacctggtcacatgcaggagcaggatggtgaagggttcttg



aaggcacttaggcaggacatttaggcaggagagaaaacctgg



aaacagaagagctgtctccaaaaatacccactggggaagcag



gttgtcatgtgggccatgaatgggacctgttctggggtaacc



acgtgcggaccgagcggccgcaggaacccctagtgatggagt



tggccactccctctctgcgcgctcgctcgctcactgaggccg



ggcgaccaaaggtcgcccgacgcccgggctttgcccgggcgg



cctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcc



tgatgcggtattttctccttacgcatctgtgcggtatttcac



accgcatacgtcaaagcaaccatagtacgcgccctgtagcgg



cgcattaagcgcggcgggtgtggtggttacgcgcagcgtgac



cgctacacttgccagcgccttagcgcccgctcctttcgcttt



cttcccttcctttctcgccacgttcgccggctttccccgtca



agctctaaatcgggggctccctttagggttccgatttagtgc



tttacggcacctcgaccccaaaaaacttgatttgggtgatgg



ttcacgtagtgggccatcgccctgatagacggtttttcgccc



tttgacgttggagtccacgttctttaatagtggactcttgtt



ccaaactggaacaacactcaactctatctcgggctattcttt



tgatttataagggattttgccgatttcggtctattggttaaa



aaatgagctgatttaacaaaaatttaacgcgaattttaacaa



aatattaacgtttacaattttatggtgcactctcagtacaat



ctgctctgatgccgcatagttaagccagccccgacacccgcc



aacacccgctgacgcgccctgacgggcttgtctgctcccggc



atccgcttacagacaagctgtgaccgtctccgggagctgcat



gtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacg



aaagggcctcgtgatacgcctatttttataggttaatgtcat



gataataatggtttcttagacgtcaggtggcacttttcgggg



aaatgtgcgcggaacccctatttgtttatttttctaaataca



ttcaaatatgtatccgctcatgagacaataaccctgataaat



gcttcaataatattgaaaaaggaagagtatgagtattcaaca



tttccgtgtcgcccttattcccttttttgcggcattttgcct



tcctgtttttgctcacccagaaacgctggtgaaagtaaaaga



tgctgaagatcagttgggtgcacgagtgggttacatcgaact



ggatctcaacagcggtaagatccttgagagttttcgccccga



agaacgttttccaatgatgagcacttttaaagttctgctatg



tggcgcggtattatcccgtattgacgccgggcaagagcaact



cggtcgccgcatacactattctcagaatgacttggttgagta



ctcaccagtcacagaaaagcatcttacggatggcatgacagt



aagagaattatgcagtgctgccataaccatgagtgataacac



tgcggccaacttacttctgacaacgatcggaggaccgaagga



gctaaccgcttttttgcacaacatgggggatcatgtaactcg



ccttgatcgttgggaaccggagctgaatgaagccataccaaa



cgacgagcgtgacaccacgatgcctgtagcaatggcaacaac



gttgcgcaaactattaactggcgaactacttactctagcttc



ccggcaacaattaatagactggatggaggcggataaagttgc



aggaccacttctgcgctcggcccttccggctggctggtttat



tgctgataaatctggagccggtgagcgtgggtctcgcggtat



cattgcagcactggggccagatggtaagccctcccgtatcgt



agttatctacacgacggggagtcaggcaactatggatgaacg



aaatagacagatcgctgagataggtgcctcactgattaagca



ttggtaactgtcagaccaagtttactcatatatactttagat



tgatttaaaacttcatttttaatttaaaaggatctaggtgaa



gatcctttttgataatctcatgaccaaaatcccttaacgtga



gttttcgttccactgagcgtcagaccccgtagaaaagatcaa



aggatcttcttgaaatcctttttttctgcgcgtaatctgctg



cttgcaaacaaaaaaaccaccgctaccagcggtggtttgttt



gccggatcaagagctaccaactctttttccgaaggtaactgg



cttcagcagagcgcagataccaaatactgttcttctagtgta



gccgtagttaggccaccacttcaagaactctgtagcaccgcc



tacatacctcgctctgctaatcctgttaccagtggctgctgc



cagtggcgataagtcgtgtcttaccgggttggactcaagacg



atagttaccggataaggcgcagcggtcgggctgaacgggggg



ttcgtgcacacagcccagcttggagcgaacgacctacaccga



actgagatacctacagcgtgagctatgagaaagcgccacgct



tcccgaagggagaaaggcggacaggtatccggtaagcggcag



ggtcggaacaggagagcgcacgagggagcttccagggggaaa



cgcctggtatctttatagtcctgtcgggtttcgccacctctg



acttgagcgtcgatttttgtgatgctcgtcaggggggcggag



cctatggaaaaacgccagcaacgcggcctttttacggttcct



ggccttttgctggccttttgctcacatgtcctgcaggcag





GENE CASSETTE
53


OF PLASMID
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


AG007 OCCURS AT
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


BP 1 THROUGH
agagggagtggccaactccatcactaggggttcctgcggccg


4645 OF SEQ ID
cacgcgttacgtaatatttattgaagtttaatattgtgtttg


NO: 28
tgatacagaagtatttgctttaattctaaataaaaattttat



gcttttattgctggtttaagaagatttggattatccttgtac



tttgaggagaagtttcttatttgaaatattttggaaacaggt



cttttaatgtggaaagatagatattaatctcctcttctatta



ctctccaagatccaacaaaagtgattataccccccaaaatat



gatggtagtatcttatactaccatcattttataggcataggg



ctcttagctgcaaataatggaactaactctaataaagcagaa



cgcaaatattgtaaatattagagagctaacaatctctgggat



ggctaaaggatggagcttggaggctacccagccagtaacaat



attccgggctccactgttgaatggagacactacaactgcctt



ggatgggcagagatattatggatgctaagccccaggtgctac



cattaggacttctaccactgtccctaacgggtggagcccatc



acatgcctatgccctcactgtaaggaaatgaagctactgttg



tatatcttgggaagcacttggattaattgttatacagttttg



ttgaagaagacccctagggtaagtagccataactgcacacta



aatttaaaattgttaatgagtttctcaaaaaaaatgttaagg



ttgttagctggtatagtatatatcttgcctgttttccaagga



cttctttgggcagtaccttgtctgtgctggcaagcaactgag



acttaatgaaagagtattggagatatgaatgaattgatgctg



tatactctcagagtgccaaacatataccaatggacaagaagg



tgaggcagagagcagacaggcattagtgacaagcaaagatat



gcagaatttcattctcagcaaatcaaaagtcctcaacctggt



tggaagaatattggcactgaatggtatcaataaggttgctag



agagggttagaggtgcacaatgtgcttccataacattttata



cttctccaatcttagcactaatcaaacatggttgaatacttt



gtttactataactcttacagagttataagatctgtgaagaca



gggacagggacaatacccatctctgtctggttcataggtggt



atgtaatagatatttttaaaaataagtgagttaatgaatgag



ggtgagaatgaaggcacagaggtattagggggaggtgggccc



cagagaatggtgccaaggtccagtggggtgactgggatcagc



tcaggcctgacgctggccactcccacctagctcctttctttc



taatctgttctcattctccttgggaaggattgaggtctctgg



aaaacagccaaacaactgttatgggaacagcaagcccaaata



aagccaagcatcagggggatctgagagctgaaagcaacttct



gttccccctccctcagctgaaggggtggggaagggctcccaa



agccataactccttttaagggatttagaaggcataaaaaggc



ccctggctgagaacttccttcttcattctgcagttggtgaat



tcgccaccatgtcagaaggggtgggcacgttccgcatggtac



ctgaagaggaacaggagctccgtgcccaactggagcagctca



caaccaaggaccatggacctgtctttggcccgtgcagccagc



tgccccgccacaccttgcagaaggccaaggatgagctgaacg



agagagaggagacccgggaggaggcagtgcgagagctgcagg



agatggtgcaggcgcaggcggcctcgggggaggagctggcgg



tggccgtggcggagagggtgcaagagaaggacagcggcttct



tcctgcgcttcatccgcgcacggaagttcaacgtgggccgtg



cctatgagctgctcagaggctatgtgaatttccggctgcagt



accctgagctctttgacagcctgtccccagaggctgtccgct



gcaccattgaagctggctaccctggtgtcctctctagtcggg



acaagtatggccgagtggtcatgctcttcaacattgagaact



ggcaaagtcaagaaatcacctttgatgagatcttgcaggcat



attgcttcatcctggagaagctgctggagaatgaggaaactc



aaatcaatggcttctgcatcattgagaacttcaagggcttta



ccatgcagcaggctgctagtctccggacttcagatctcagga



agatggtggacatgctccaggattccttcccagcccggttca



aagccatccacttcatccaccagccatggtacttcaccacga



cctacaatgtggtcaagcccttcttgaagagcaagctgcttg



agagggtctttgtccacggggatgacctttctggtttctacc



aggagatcgatgagaacatcctgccctctgacttcgggggca



cgctgcccaagtatgatggcaaggccgttgctgagcagctct



ttggcccccaggcccaagctgagaacacagccttctgaggat



ctaccggtcgacctgcagaagcttgcctcgagcagcgctgct



cgagagatctggatcataatcagccataccacatttgtagag



gttttacttgctttaaaaaacctcccacacctccccctgaac



ctgaaacataaaatgaatgcaattgttgttgttaacttgttt



attgcagcttataatggttacaaataaagcaatagcatcaca



aatttcacaaataaagcatttttttcactgcattctagttgt



ggtttgtccaaactcatcaatgtatcttatcatgtctggtaa



ccattctccaggttgagccagaccaatttgatggtagattta



gcaaataaaaatacaggacacccagttaaatgtgaatttccg



atgaacagcaaatacttttttagtattaaaaaagttcacatt



taggctcacgcctgtaatcccagcactttgggaggccgaggc



aggcagatcacctgaggtcaggagttcgagaccagcctggcc



aacatggtgaaaccccatctccactaaaaataccaaaaatta



gccaggcgtgctggtgggcacctgtagttccagctactcagg



aggctaaggcaggagaattgcttgaacctgggaggcagaggt



tgcagtgagctgagatcgcaccattgcactctagcctgggcg



acaagaacaaaactccatctcaaaaaaaaaaaaaaaaaaaaa



gttcacatttaactgggcattctgtatttaattggtaatctg



agatggcagggaacagcatcagcatggtgtgagggataggca



ttttttcattgtgtacagcttgtaaatcagtatttttaaaac



tcaaagttaatggcttgggcatatttagaaaagagttgccgc



acggacttgaaccctgtattcctaaaatctaggatcttgttc



tgatggtctgcacaactggctgggggtgtccagccactgtcc



ctcttgcctgggctccccagggcagttctgtcagcctctcca



tttccattcctgttccagcaaaacccaactgatagcacagca



gcatttcagcctgtctacctctgtgcccacatacctggatgt



ctaccagccagaaaggtggcttagatttggttcctgtgggtg



gattatggcccccagaacttccctgtgcttgctgggggtgtg



gagtggaaagagcaggaaatgggggaccctccgatactctat



gggggtcctccaagtctctttgtgcaagttagggtaataatc



aatatggagctaagaaagagaaggggaactatgctttagaac



aggacactgtgccaggagcattgcagaaattatatggttttc



acgacagttctttttggtaggtactgttattatcctcagttt



gcagatgaggaaactgagacccagaaaggttaaataacttgc



tagggtcacacaagtcataactgacaaagcctgattcaaacc



caggtctccctaacctttaaggtttctatgacgccagctctc



ctagggagtttgtcttcagatgtcttggctctaggtgtcaaa



aaaagacttggtgtcaggcaggcataggttcaagtcccaact



ctgtcacttaccaactgtgactaggtgattgaactgaccatg



gaacctggtcacatgcaggagcaggatggtgaagggttcttg



aaggcacttaggcaggacatttaggcaggagagaaaacctgg



aaacagaagagctgtctccaaaaatacccactggggaagcag



gttgtcatgtgggccatgaatgggacctgttctggggtaacc



acgtgcggaccgagcggccgcaggaacccctagtgatggagt



tggccactccctctctgcgcgctcgctcgctcactgaggccg



ggcgaccaaaggtcgcccgacgcccgggctttgcccgggcgg



cctcagtgagcgagcgagcgcgcag










Plasmid TM039 Composition








5′ ITR
 2



occurs at bp 1 through bp 119 of SEQ ID



NO: 29





CVM ENHANCER
22


AND CBA
occurs at bp 134 through bp 1749 of SEQ ID


PROMOTER
NO: 29


GENBANK


ACCESSION


DD215332 FROM


BP 1-BP 1616)





Added Kozak
 5



occurs at bp 1763 through bp 1768 of SEQ



ID NO: 29





HUMAN RLBP1
 6


GENE CDS
occurs at bp 1769 through bp 2722 of SEQ



ID NO: 29





SV40 POLYA
 8



occurs at bp 2781 through bp 3016 of SEQ



ID NO: 29





REVERSE
23


COMPLEMENT OF
occurs at bp 3032 through bp 4534 of SEQ


RLBP1 INTRONIC
ID NO: 29


SEQUENCE AS


STUFFER


SEQUENCE


(NT 010274.17)





3′ ITR
 9



occurs at bp 4573 through bp 4702 of SEQ



ID NO: 29





AMP BACTERIAL
15


BACKBONE
occurs at bp 4703 through bp 7321 of SEQ



ID NO: 29





PLASMID TM039
29


SEQUENCE
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc



gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag



agagggagtggccaactccatcactaggggttcctgcggccg



cacgcgtactagttattaatagtaatcaattacggggtcatt



agttcatagcccatatatggagttccgcgttacataacttac



ggtaaatggcccgcctggctgaccgcccaacgacccccgccc



attgacgtcaataatgacgtatgttcccatagtaacgccaat



agggactttccattgacgtcaatgggtggagtatttacggta



aactgcccacttggcagtacatcaagtgtatcatatgccaag



tacgccccctattgacgtcaatgacggtaaatggcccgcctg



gcattatgcccagtacatgaccttatgggactttcctacttg



gcagtacatctacgtattagtcatcgctattaccatggtcga



ggtgagccccacgttctgcttcactctccccatctccccccc



ctccccacccccaattttgtatttatttattttttaattatt



ttgtgcagcgatgggggcggggggggggggggggcgcgcgcc



aggcggggcggggcggggcgaggggcggggcggggcgaggcg



gagaggtgcggcggcagccaatcagagcggcgcgctccgaaa



gtttccttttatggcgaggcggcggcggcggcggccctataa



aaagcgaagcgcgcggcgggcggggagtcgctgcgacgctgc



cttcgccccgtgccccgctccgccgccgcctcgcgccgcccg



ccccggctctgactgaccgcgttactcccacaggtgagcggg



cgggacggcccttctcctccgggctgtaattagcgcttggtt



taatgacggcttgtttcttttctgtggctgcgtgaaagcctt



gaggggctccgggagggccctttgtgcggggggagcggctcg



gggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgc



ggctccgcgctgcccggcggctgtgagcgctgcgggcgcggc



gcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcg



gccgggggcggtgccccgcggtgcggggggggctgcgagggg



aacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagca



gggggtgtgggcgcgtcggtcgggctgcaaccccccctgcac



ccccctccccgagttgctgagcacggcccggcttcgggtgcg



gggctccgtacggggcgtggcgcggggctcgccgtgccgggc



ggggggtggcggcaggtgggggtgccgggcggggcggggccg



cctcgggccggggagggctcgggggaggggcgcggcggcccc



cggagcgccggcggctgtcgaggcgcggcgagccgcagccat



tgccttttatggtaatcgtgcgagagggcgcagggacttcct



ttgtcccaaatctgtgcggagccgaaatctgggaggcgccgc



cgcaccccctctagcgggcgcggggcgaagcggtgcggcgcc



ggcaggaaggaaatgggcggggagggccttcgtgcgtcgccg



cgccgccgtccccttctccctctccagcctcggggctgtccg



cggggggacggctgccttcgggggggacggggcagggcgggg



ttcggcttctggcgtgtgaccggcggcatcgattgaattcgc



caccatgtcagaaggggtgggcacgttccgcatggtacctga



agaggaacaggagctccgtgcccaactggagcagctcacaac



caaggaccatggacctgtctttggcccgtgcagccagctgcc



ccgccacaccttgcagaaggccaaggatgagctgaacgagag



agaggagacccgggaggaggcagtgcgagagctgcaggagat



ggtgcaggcgcaggcggcctcgggggaggagctggcggtggc



cgtggcggagagggtgcaagagaaggacagcggcttcttcct



gcgcttcatccgcgcacggaagttcaacgtgggccgtgccta



tgagctgctcagaggctatgtgaatttccggctgcagtaccc



tgagctctttgacagcctgtccccagaggctgtccgctgcac



cattgaagctggctaccctggtgtcctctctagtcgggacaa



gtatggccgagtggtcatgctcttcaacattgagaactggca



aagtcaagaaatcacctttgatgagatcttgcaggcatattg



cttcatcctggagaagctgctggagaatgaggaaactcaaat



caatggcttctgcatcattgagaacttcaagggctttaccat



gcagcaggctgctagtctccggacttcagatctcaggaagat



ggtggacatgctccaggattccttcccagcccggttcaaagc



catccacttcatccaccagccatggtacttcaccacgaccta



caatgtggtcaagcccttcttgaagagcaagctgcttgagag



ggtctttgtccacggggatgacctttctggtttctaccagga



gatcgatgagaacatcctgccctctgacttcgggggcacgct



gcccaagtatgatggcaaggccgttgctgagcagctctttgg



cccccaggcccaagctgagaacacagccttctgaggatcgta



ccggtcgacctgcagaagcttgcctcgagcagcgctgctcga



gagatctggatcataatcagccataccacatttgtagaggtt



ttacttgctttaaaaaacctcccacacctccccctgaacctg



aaacataaaatgaatgcaattgttgttgttaacttgtttatt



gcagcttataatggttacaaataaagcaatagcatcacaaat



ttcacaaataaagcatttttttcactgcattctagttgtggt



ttgtccaaactcatcaatgtatcttatcatgtctggtactag



ggttaccccagaacaggtcccattcatggcccacatgacaac



ctgcttccccagtgggtatttttggagacagctcttctgttt



ccaggttttctctcctgcctaaatgtcctgcctaagtgcctt



caagaacccttcaccatcctgctcctgcatgtgaccaggttc



catggtcagttcaatcacctagtcacagttggtaagtgacag



agttgggacttgaacctatgcctgcctgacaccaagtctttt



tttgacacctagagccaagacatctgaagacaaactccctag



gagagctggcgtcatagaaaccttaaaggttagggagacctg



ggtttgaatcaggctttgtcagttatgacttgtgtgacccta



gcaagttatttaacctttctgggtctcagtttcctcatctgc



aaactgaggataataacagtacctaccaaaaagaactgtcgt



gaaaaccatataatttctgcaatgctcctggcacagtgtcct



gttctaaagcatagttccccttctctttcttagctccatatt



gattattaccctaacttgcacaaagagacttggaggaccccc



atagagtatcggagggtcccccatttcctgctctttccactc



cacacccccagcaagcacagggaagttctgggggccataatc



cacccacaggaaccaaatctaagccacctttctggctggtag



acatccaggtatgtgggcacagaggtagacaggctgaaatgc



tgctgtgctatcagttgggttttgctggaacaggaatggaaa



tggagaggctgacagaactgccctggggagcccaggcaagag



ggacagtggctggacacccccagccagttgtgcagaccatca



gaacaagatcctagattttaggaatacagggttcaagtccgt



gcggcaactcttttctaaatatgcccaagccattaactttga



gttttaaaaatactgatttacaagctgtacacaatgaaaaaa



tgcctatccctcacaccatgctgatgctgttccctgccatct



cagattaccaattaaatacagaatgcccagttaaatgtgaac



tttttttttttttttttttttgagatggagttttgttcttgt



cgcccaggctagagtgcaatggtgcgatctcagctcactgca



acctctgcctcccaggttcaagcaattctcctgccttagcct



cctgagtagctggaactacaggtgcccaccagcacgcctggc



taatttttggtatttttagtggagatggggtttcaccatgtt



ggccaggctggtctcgaactcctgacctcaggtgatctgcct



gcctcggcctcccaaagtgctgggattacaggcgtgagccta



aatgtgaacttttttaatactaaaaaagtatttgctgttcat



cggaaattcacatttaactgggtgtcctgtatttttatttgc



taaatctaccatcaaattggtctggctcaacctggagaatgg



ttaccctaggtaaccacgtgcggaccgagcggccgcaggaac



ccctagtgatggagttggccactccctctctgcgcgctcgct



cgctcactgaggccgggcgaccaaaggtcgcccgacgcccgg



gctttgcccgggcggcctcagtgagcgagcgagcgcgcagct



gcctgcaggggcgcctgatgcggtattttctccttacgcatc



tgtgcggtatttcacaccgcatacgtcaaagcaaccatagta



cgcgccctgtagcggcgcattaagcgcggcgggtgtggtggt



tacgcgcagcgtgaccgctacacttgccagcgccttagcgcc



cgctcctttcgctttcttcccttcctttctcgccacgttcgc



cggctttccccgtcaagctctaaatcgggggctccctttagg



gttccgatttagtgctttacggcacctcgaccccaaaaaact



tgatttgggtgatggttcacgtagtgggccatcgccctgata



gacggtttttcgccctttgacgttggagtccacgttctttaa



tagtggactcttgttccaaactggaacaacactcaactctat



ctcgggctattcttttgatttataagggattttgccgatttc



ggtctattggttaaaaaatgagctgatttaacaaaaatttaa



cgcgaattttaacaaaatattaacgtttacaattttatggtg



cactctcagtacaatctgctctgatgccgcatagttaagcca



gccccgacacccgccaacacccgctgacgcgccctgacgggc



ttgtctgctcccggcatccgcttacagacaagctgtgaccgt



ctccgggagctgcatgtgtcagaggttttcaccgtcatcacc



gaaacgcgcgagacgaaagggcctcgtgatacgcctattttt



ataggttaatgtcatgataataatggtttcttagacgtcagg



tggcacttttcggggaaatgtgcgcggaacccctatttgttt



atttttctaaatacattcaaatatgtatccgctcatgagaca



ataaccctgataaatgcttcaataatattgaaaaaggaagag



tatgagtattcaacatttccgtgtcgcccttattcccttttt



tgcggcattttgccttcctgtttttgctcacccagaaacgct



ggtgaaagtaaaagatgctgaagatcagttgggtgcacgagt



gggttacatcgaactggatctcaacagcggtaagatccttga



gagttttcgccccgaagaacgttttccaatgatgagcacttt



taaagttctgctatgtggcgcggtattatcccgtattgacgc



cgggcaagagcaactcggtcgccgcatacactattctcagaa



tgacttggttgagtactcaccagtcacagaaaagcatcttac



ggatggcatgacagtaagagaattatgcagtgctgccataac



catgagtgataacactgcggccaacttacttctgacaacgat



cggaggaccgaaggagctaaccgcttttttgcacaacatggg



ggatcatgtaactcgccttgatcgttgggaaccggagctgaa



tgaagccataccaaacgacgagcgtgacaccacgatgcctgt



agcaatggcaacaacgttgcgcaaactattaactggcgaact



acttactctagcttcccggcaacaattaatagactggatgga



ggcggataaagttgcaggaccacttctgcgctcggcccttcc



ggctggctggtttattgctgataaatctggagccggtgagcg



tgggtctcgcggtatcattgcagcactggggccagatggtaa



gccctcccgtatcgtagttatctacacgacggggagtcaggc



aactatggatgaacgaaatagacagatcgctgagataggtgc



ctcactgattaagcattggtaactgtcagaccaagtttactc



atatatactttagattgatttaaaacttcatttttaatttaa



aaggatctaggtgaagatcctttttgataatctcatgaccaa



aatcccttaacgtgagttttcgttccactgagcgtcagaccc



cgtagaaaagatcaaaggatcttcttgaaatcctttttttct



gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacc



agcggtggtttgtttgccggatcaagagctaccaactctttt



tccgaaggtaactggcttcagcagagcgcagataccaaatac



tgttcttctagtgtagccgtagttaggccaccacttcaagaa



ctctgtagcaccgcctacatacctcgctctgctaatcctgtt



accagtggctgctgccagtggcgataagtcgtgtcttaccgg



gttggactcaagacgatagttaccggataaggcgcagcggtc



gggctgaacggggggttcgtgcacacagcccagcttggagcg



aacgacctacaccgaactgagatacctacagcgtgagctatg



agaaagcgccacgcttcccgaagggagaaaggcggacaggta



tccggtaagcggcagggtcggaacaggagagcgcacgaggga



gcttccagggggaaacgcctggtatctttatagtcctgtcgg



gtttcgccacctctgacttgagcgtcgatttttgtgatgctc



gtcaggggggcggagcctatggaaaaacgccagcaacgcggc



ctttttacggttcctggccttttgctggccttttgctcacat



gtcctgcaggcag





GENE CASSETTE
54


OF PLASMID
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


TM039 OCCURS AT
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


BP 1 THROUGH
agagggagtggccaactccatcactaggggttcctgcggccg


4702 OF SEQ ID
cacgcgtactagttattaatagtaatcaattacggggtcatt


NO: 29
agttcatagcccatatatggagttccgcgttacataacttac



ggtaaatggcccgcctggctgaccgcccaacgacccccgccc



attgacgtcaataatgacgtatgttcccatagtaacgccaat



agggactttccattgacgtcaatgggtggagtatttacggta



aactgcccacttggcagtacatcaagtgtatcatatgccaag



tacgccccctattgacgtcaatgacggtaaatggcccgcctg



gcattatgcccagtacatgaccttatgggactttcctacttg



gcagtacatctacgtattagtcatcgctattaccatggtcga



ggtgagccccacgttctgcttcactctccccatctccccccc



ctccccacccccaattttgtatttatttattttttaattatt



ttgtgcagcgatgggggcggggggggggggggggcgcgcgcc



aggcggggcggggcggggcgaggggcggggcggggcgaggcg



gagaggtgcggcggcagccaatcagagcggcgcgctccgaaa



gtttccttttatggcgaggcggcggcggcggcggccctataa



aaagcgaagcgcgcggcgggcggggagtcgctgcgacgctgc



cttcgccccgtgccccgctccgccgccgcctcgcgccgcccg



ccccggctctgactgaccgcgttactcccacaggtgagcggg



cgggacggcccttctcctccgggctgtaattagcgcttggtt



taatgacggcttgtttcttttctgtggctgcgtgaaagcctt



gaggggctccgggagggccctttgtgcggggggagcggctcg



gggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgc



ggctccgcgctgcccggcggctgtgagcgctgcgggcgcggc



gcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcg



gccgggggcggtgccccgcggtgcggggggggctgcgagggg



aacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagca



gggggtgtgggcgcgtcggtcgggctgcaaccccccctgcac



ccccctccccgagttgctgagcacggcccggcttcgggtgcg



gggctccgtacggggcgtggcgcggggctcgccgtgccgggc



ggggggtggcggcaggtgggggtgccgggcggggcggggccg



cctcgggccggggagggctcgggggaggggcgcggcggcccc



cggagcgccggcggctgtcgaggcgcggcgagccgcagccat



tgccttttatggtaatcgtgcgagagggcgcagggacttcct



ttgtcccaaatctgtgcggagccgaaatctgggaggcgccgc



cgcaccccctctagcgggcgcggggcgaagcggtgcggcgcc



ggcaggaaggaaatgggcggggagggccttcgtgcgtcgccg



cgccgccgtccccttctccctctccagcctcggggctgtccg



cggggggacggctgccttcgggggggacggggcagggcgggg



ttcggcttctggcgtgtgaccggcggcatcgattgaattcgc



caccatgtcagaaggggtgggcacgttccgcatggtacctga



agaggaacaggagctccgtgcccaactggagcagctcacaac



caaggaccatggacctgtctttggcccgtgcagccagctgcc



ccgccacaccttgcagaaggccaaggatgagctgaacgagag



agaggagacccgggaggaggcagtgcgagagctgcaggagat



ggtgcaggcgcaggcggcctcgggggaggagctggcggtggc



cgtggcggagagggtgcaagagaaggacagcggcttcttcct



gcgcttcatccgcgcacggaagttcaacgtgggccgtgccta



tgagctgctcagaggctatgtgaatttccggctgcagtaccc



tgagctctttgacagcctgtccccagaggctgtccgctgcac



cattgaagctggctaccctggtgtcctctctagtcgggacaa



gtatggccgagtggtcatgctcttcaacattgagaactggca



aagtcaagaaatcacctttgatgagatcttgcaggcatattg



cttcatcctggagaagctgctggagaatgaggaaactcaaat



caatggcttctgcatcattgagaacttcaagggctttaccat



gcagcaggctgctagtctccggacttcagatctcaggaagat



ggtggacatgctccaggattccttcccagcccggttcaaagc



catccacttcatccaccagccatggtacttcaccacgaccta



caatgtggtcaagcccttcttgaagagcaagctgcttgagag



ggtctttgtccacggggatgacctttctggtttctaccagga



gatcgatgagaacatcctgccctctgacttcgggggcacgct



gcccaagtatgatggcaaggccgttgctgagcagctctttgg



cccccaggcccaagctgagaacacagccttctgaggatcgta



ccggtcgacctgcagaagcttgcctcgagcagcgctgctcga



gagatctggatcataatcagccataccacatttgtagaggtt



ttacttgctttaaaaaacctcccacacctccccctgaacctg



aaacataaaatgaatgcaattgttgttgttaacttgtttatt



gcagcttataatggttacaaataaagcaatagcatcacaaat



ttcacaaataaagcatttttttcactgcattctagttgtggt



ttgtccaaactcatcaatgtatcttatcatgtctggtactag



ggttaccccagaacaggtcccattcatggcccacatgacaac



ctgcttccccagtgggtatttttggagacagctcttctgttt



ccaggttttctctcctgcctaaatgtcctgcctaagtgcctt



caagaacccttcaccatcctgctcctgcatgtgaccaggttc



catggtcagttcaatcacctagtcacagttggtaagtgacag



agttgggacttgaacctatgcctgcctgacaccaagtctttt



tttgacacctagagccaagacatctgaagacaaactccctag



gagagctggcgtcatagaaaccttaaaggttagggagacctg



ggtttgaatcaggctttgtcagttatgacttgtgtgacccta



gcaagttatttaacctttctgggtctcagtttcctcatctgc



aaactgaggataataacagtacctaccaaaaagaactgtcgt



gaaaaccatataatttctgcaatgctcctggcacagtgtcct



gttctaaagcatagttccccttctctttcttagctccatatt



gattattaccctaacttgcacaaagagacttggaggaccccc



atagagtatcggagggtcccccatttcctgctctttccactc



cacacccccagcaagcacagggaagttctgggggccataatc



cacccacaggaaccaaatctaagccacctttctggctggtag



acatccaggtatgtgggcacagaggtagacaggctgaaatgc



tgctgtgctatcagttgggttttgctggaacaggaatggaaa



tggagaggctgacagaactgccctggggagcccaggcaagag



ggacagtggctggacacccccagccagttgtgcagaccatca



gaacaagatcctagattttaggaatacagggttcaagtccgt



gcggcaactcttttctaaatatgcccaagccattaactttga



gttttaaaaatactgatttacaagctgtacacaatgaaaaaa



tgcctatccctcacaccatgctgatgctgttccctgccatct



cagattaccaattaaatacagaatgcccagttaaatgtgaac



tttttttttttttttttttttgagatggagttttgttcttgt



cgcccaggctagagtgcaatggtgcgatctcagctcactgca



acctctgcctcccaggttcaagcaattctcctgccttagcct



cctgagtagctggaactacaggtgcccaccagcacgcctggc



taatttttggtatttttagtggagatggggtttcaccatgtt



ggccaggctggtctcgaactcctgacctcaggtgatctgcct



gcctcggcctcccaaagtgctgggattacaggcgtgagccta



aatgtgaacttttttaatactaaaaaagtatttgctgttcat



cggaaattcacatttaactgggtgtcctgtatttttatttgc



taaatctaccatcaaattggtctggctcaacctggagaatgg



ttaccctaggtaaccacgtgcggaccgagcggccgcaggaac



ccctagtgatggagttggccactccctctctgcgcgctcgct



cgctcactgaggccgggcgaccaaaggtcgcccgacgcccgg



gctttgcccgggcggcctcagtgagcgagcgagcgcgcag










Plasmid TM040 Composition








5′ ITR
 2



occurs at bp 1 through bp 119 of SEQ ID



NO: 30





Human RLBP1
 3


Promoter(short)
occurs at bp 134 through bp 723 of SEQ ID



NO: 30





Modified SV40
 4


intron
occurs at bp 738 through bp 920 of SEQ ID



NO: 30





Added Kozak
 5



occurs at bp 934 through bp 939 of SEQ ID



NO: 30





HUMAN RLBP1
 6


GENE CDS
occurs at bp 940 through bp 1893 of SEQ ID



NO: 30





SV40 POLYA
 8



occurs at bp 1952 through bp 2187 of SEQ



ID NO: 30





REVERSE
23


COMPLEMENT OF
occurs at bp 2203 through bp 3705 of SEQ


RLBP1 INTRONIC
ID NO: 30


SEQUENCE AS


STUFFER


SEQUENCE


(NT 010274.17)





3′ ITR
 9



occurs at bp 3744 through bp 3873 of SEQ



ID NO: 30





AMP BACTERIAL
15


BACKBONE
occurs at bp 3874 through bp 6492 of SEQ



ID NO: 30





TM040 plasmid
30


sequence
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc



gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag



agagggagtggccaactccatcactaggggttcctgcggccg



cacgcgtttgtcctctccctgcttggccttaaccagccacat



ttctcaactgaccccactcactgcagaggtgaaaactaccat



gccaggtcctgctggctgggggaggggtgggcaataggcctg



gatttgccagagctgccactgtagatgtagtcatatttacga



tttcccttcacctcttattaccctggtggtggtggtgggggg



gggggggtgctctctcagcaaccccaccccgggatcttgagg



agaaagagggcagagaaaagagggaatgggactggcccagat



cccagccccacagccgggcttccacatggccgagcaggaact



ccagagcaggagcacacaaaggagggctttgatgcgcctcca



gccaggcccaggcctctcccctctcccctttctctctgggtc



ttcctttgccccactgagggcctcctgtgagcccgatttaac



ggaaactgtgggcggtgagaagttccttatgacacactaatc



ccaacctgctgaccggaccacgcctccagcggagggaacctc



tagagctccaggacattcaggtaccaggtagccccaaggagg



agctgccgaatcgatggatcgggaactgaaaaaccagaaagt



taactggtaagtttagtctttttgtcttttatttcaggtccc



ggatccggtggtggtgcaaatcaaagaactgctcctcagtgg



atgttgcctttacttctaggcctgtacggaagtgttacttct



gctctaaaagctgcggaattgtacccgccccgggatccatcg



attgaattcgccaccatgtcagaaggggtgggcacgttccgc



atggtacctgaagaggaacaggagctccgtgcccaactggag



cagctcacaaccaaggaccatggacctgtctttggcccgtgc



agccagctgccccgccacaccttgcagaaggccaaggatgag



ctgaacgagagagaggagacccgggaggaggcagtgcgagag



ctgcaggagatggtgcaggcgcaggcggcctcgggggaggag



ctggcggtggccgtggcggagagggtgcaagagaaggacagc



ggcttcttcctgcgcttcatccgcgcacggaagttcaacgtg



ggccgtgcctatgagctgctcagaggctatgtgaatttccgg



ctgcagtaccctgagctctttgacagcctgtccccagaggct



gtccgctgcaccattgaagctggctaccctggtgtcctctct



agtcgggacaagtatggccgagtggtcatgctcttcaacatt



gagaactggcaaagtcaagaaatcacctttgatgagatcttg



caggcatattgcttcatcctggagaagctgctggagaatgag



gaaactcaaatcaatggcttctgcatcattgagaacttcaag



ggctttaccatgcagcaggctgctagtctccggacttcagat



ctcaggaagatggtggacatgctccaggattccttcccagcc



cggttcaaagccatccacttcatccaccagccatggtacttc



accacgacctacaatgtggtcaagcccttcttgaagagcaag



ctgcttgagagggtctttgtccacggggatgacctttctggt



ttctaccaggagatcgatgagaacatcctgccctctgacttc



gggggcacgctgcccaagtatgatggcaaggccgttgctgag



cagctctttggcccccaggcccaagctgagaacacagccttc



tgaggatcgtaccggtcgacctgcagaagcttgcctcgagca



gcgctgctcgagagatctggatcataatcagccataccacat



ttgtagaggttttacttgctttaaaaaacctcccacacctcc



ccctgaacctgaaacataaaatgaatgcaattgttgttgtta



acttgtttattgcagcttataatggttacaaataaagcaata



gcatcacaaatttcacaaataaagcatttttttcactgcatt



ctagttgtggtttgtccaaactcatcaatgtatcttatcatg



tctggtactagggttaccccagaacaggtcccattcatggcc



cacatgacaacctgcttccccagtgggtatttttggagacag



ctcttctgtttccaggttttctctcctgcctaaatgtcctgc



ctaagtgccttcaagaacccttcaccatcctgctcctgcatg



tgaccaggttccatggtcagttcaatcacctagtcacagttg



gtaagtgacagagttgggacttgaacctatgcctgcctgaca



ccaagtctttttttgacacctagagccaagacatctgaagac



aaactccctaggagagctggcgtcatagaaaccttaaaggtt



agggagacctgggtttgaatcaggctttgtcagttatgactt



gtgtgaccctagcaagttatttaacctttctgggtctcagtt



tcctcatctgcaaactgaggataataacagtacctaccaaaa



agaactgtcgtgaaaaccatataatttctgcaatgctcctgg



cacagtgtcctgttctaaagcatagttccccttctctttctt



agctccatattgattattaccctaacttgcacaaagagactt



ggaggacccccatagagtatcggagggtcccccatttcctgc



tctttccactccacacccccagcaagcacagggaagttctgg



gggccataatccacccacaggaaccaaatctaagccaccttt



ctggctggtagacatccaggtatgtgggcacagaggtagaca



ggctgaaatgctgctgtgctatcagttgggttttgctggaac



aggaatggaaatggagaggctgacagaactgccctggggagc



ccaggcaagagggacagtggctggacacccccagccagttgt



gcagaccatcagaacaagatcctagattttaggaatacaggg



ttcaagtccgtgcggcaactcttttctaaatatgcccaagcc



attaactttgagttttaaaaatactgatttacaagctgtaca



caatgaaaaaatgcctatccctcacaccatgctgatgctgtt



ccctgccatctcagattaccaattaaatacagaatgcccagt



taaatgtgaactttttttttttttttttttttgagatggagt



tttgttcttgtcgcccaggctagagtgcaatggtgcgatctc



agctcactgcaacctctgcctcccaggttcaagcaattctcc



tgccttagcctcctgagtagctggaactacaggtgcccacca



gcacgcctggctaatttttggtatttttagtggagatggggt



ttcaccatgttggccaggctggtctcgaactcctgacctcag



gtgatctgcctgcctcggcctcccaaagtgctgggattacag



gcgtgagcctaaatgtgaacttttttaatactaaaaaagtat



ttgctgttcatcggaaattcacatttaactgggtgtcctgta



tttttatttgctaaatctaccatcaaattggtctggctcaac



ctggagaatggttaccctaggtaaccacgtgcggaccgagcg



gccgcaggaacccctagtgatggagttggccactccctctct



gcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc



ccgacgcccgggctttgcccgggcggcctcagtgagcgagcg



agcgcgcagctgcctgcaggggcgcctgatgcggtattttct



ccttacgcatctgtgcggtatttcacaccgcatacgtcaaag



caaccatagtacgcgccctgtagcggcgcattaagcgcggcg



ggtgtggtggttacgcgcagcgtgaccgctacacttgccagc



gccttagcgcccgctcctttcgctttcttcccttcctttctc



gccacgttcgccggctttccccgtcaagctctaaatcggggg



ctccctttagggttccgatttagtgctttacggcacctcgac



cccaaaaaacttgatttgggtgatggttcacgtagtgggcca



tcgccctgatagacggtttttcgccctttgacgttggagtcc



acgttctttaatagtggactcttgttccaaactggaacaaca



ctcaactctatctcgggctattcttttgatttataagggatt



ttgccgatttcggtctattggttaaaaaatgagctgatttaa



caaaaatttaacgcgaattttaacaaaatattaacgtttaca



attttatggtgcactctcagtacaatctgctctgatgccgca



tagttaagccagccccgacacccgccaacacccgctgacgcg



ccctgacgggcttgtctgctcccggcatccgcttacagacaa



gctgtgaccgtctccgggagctgcatgtgtcagaggttttca



ccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgata



cgcctatttttataggttaatgtcatgataataatggtttct



tagacgtcaggtggcacttttcggggaaatgtgcgcggaacc



cctatttgtttatttttctaaatacattcaaatatgtatccg



ctcatgagacaataaccctgataaatgcttcaataatattga



aaaaggaagagtatgagtattcaacatttccgtgtcgccctt



attcccttttttgcggcattttgccttcctgtttttgctcac



ccagaaacgctggtgaaagtaaaagatgctgaagatcagttg



ggtgcacgagtgggttacatcgaactggatctcaacagcggt



aagatccttgagagttttcgccccgaagaacgttttccaatg



atgagcacttttaaagttctgctatgtggcgcggtattatcc



cgtattgacgccgggcaagagcaactcggtcgccgcatacac



tattctcagaatgacttggttgagtactcaccagtcacagaa



aagcatcttacggatggcatgacagtaagagaattatgcagt



gctgccataaccatgagtgataacactgcggccaacttactt



ctgacaacgatcggaggaccgaaggagctaaccgcttttttg



cacaacatgggggatcatgtaactcgccttgatcgttgggaa



ccggagctgaatgaagccataccaaacgacgagcgtgacacc



acgatgcctgtagcaatggcaacaacgttgcgcaaactatta



actggcgaactacttactctagcttcccggcaacaattaata



gactggatggaggcggataaagttgcaggaccacttctgcgc



tcggcccttccggctggctggtttattgctgataaatctgga



gccggtgagcgtgggtctcgcggtatcattgcagcactgggg



ccagatggtaagccctcccgtatcgtagttatctacacgacg



gggagtcaggcaactatggatgaacgaaatagacagatcgct



gagataggtgcctcactgattaagcattggtaactgtcagac



caagtttactcatatatactttagattgatttaaaacttcat



ttttaatttaaaaggatctaggtgaagatcctttttgataat



ctcatgaccaaaatcccttaacgtgagttttcgttccactga



gcgtcagaccccgtagaaaagatcaaaggatcttcttgaaat



cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaa



ccaccgctaccagcggtggtttgtttgccggatcaagagcta



ccaactctttttccgaaggtaactggcttcagcagagcgcag



ataccaaatactgttcttctagtgtagccgtagttaggccac



cacttcaagaactctgtagcaccgcctacatacctcgctctg



ctaatcctgttaccagtggctgctgccagtggcgataagtcg



tgtcttaccgggttggactcaagacgatagttaccggataag



gcgcagcggtcgggctgaacggggggttcgtgcacacagccc



agcttggagcgaacgacctacaccgaactgagatacctacag



cgtgagctatgagaaagcgccacgcttcccgaagggagaaag



gcggacaggtatccggtaagcggcagggtcggaacaggagag



cgcacgagggagcttccagggggaaacgcctggtatctttat



agtcctgtcgggtttcgccacctctgacttgagcgtcgattt



ttgtgatgctcgtcaggggggcggagcctatggaaaaacgcc



agcaacgcggcctttttacggttcctggccttttgctggcct



tttgctcacatgtcctgcaggcag





GENE CASSETTE
55


OF PLASMID
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


TM040 OCCURS AT
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


BP 1 THROUGH
agagggagtggccaactccatcactaggggttcctgcggccg


3873 OF SEQ ID
cacgcgtttgtcctctccctgcttggccttaaccagccacat


NO: 30
ttctcaactgaccccactcactgcagaggtgaaaactaccat



gccaggtcctgctggctgggggaggggtgggcaataggcctg



gatttgccagagctgccactgtagatgtagtcatatttacga



tttcccttcacctcttattaccctggtggtggtggtgggggg



gggggggtgctctctcagcaaccccaccccgggatcttgagg



agaaagagggcagagaaaagagggaatgggactggcccagat



cccagccccacagccgggcttccacatggccgagcaggaact



ccagagcaggagcacacaaaggagggctttgatgcgcctcca



gccaggcccaggcctctcccctctcccctttctctctgggtc



ttcctttgccccactgagggcctcctgtgagcccgatttaac



ggaaactgtgggcggtgagaagttccttatgacacactaatc



ccaacctgctgaccggaccacgcctccagcggagggaacctc



tagagctccaggacattcaggtaccaggtagccccaaggagg



agctgccgaatcgatggatcgggaactgaaaaaccagaaagt



taactggtaagtttagtctttttgtcttttatttcaggtccc



ggatccggtggtggtgcaaatcaaagaactgctcctcagtgg



atgttgcctttacttctaggcctgtacggaagtgttacttct



gctctaaaagctgcggaattgtacccgccccgggatccatcg



attgaattcgccaccatgtcagaaggggtgggcacgttccgc



atggtacctgaagaggaacaggagctccgtgcccaactggag



cagctcacaaccaaggaccatggacctgtctttggcccgtgc



agccagctgccccgccacaccttgcagaaggccaaggatgag



ctgaacgagagagaggagacccgggaggaggcagtgcgagag



ctgcaggagatggtgcaggcgcaggcggcctcgggggaggag



ctggcggtggccgtggcggagagggtgcaagagaaggacagc



ggcttcttcctgcgcttcatccgcgcacggaagttcaacgtg



ggccgtgcctatgagctgctcagaggctatgtgaatttccgg



ctgcagtaccctgagctctttgacagcctgtccccagaggct



gtccgctgcaccattgaagctggctaccctggtgtcctctct



agtcgggacaagtatggccgagtggtcatgctcttcaacatt



gagaactggcaaagtcaagaaatcacctttgatgagatcttg



caggcatattgcttcatcctggagaagctgctggagaatgag



gaaactcaaatcaatggcttctgcatcattgagaacttcaag



ggctttaccatgcagcaggctgctagtctccggacttcagat



ctcaggaagatggtggacatgctccaggattccttcccagcc



cggttcaaagccatccacttcatccaccagccatggtacttc



accacgacctacaatgtggtcaagcccttcttgaagagcaag



ctgcttgagagggtctttgtccacggggatgacctttctggt



ttctaccaggagatcgatgagaacatcctgccctctgacttc



gggggcacgctgcccaagtatgatggcaaggccgttgctgag



cagctctttggcccccaggcccaagctgagaacacagccttc



tgaggatcgtaccggtcgacctgcagaagcttgcctcgagca



gcgctgctcgagagatctggatcataatcagccataccacat



ttgtagaggttttacttgctttaaaaaacctcccacacctcc



ccctgaacctgaaacataaaatgaatgcaattgttgttgtta



acttgtttattgcagcttataatggttacaaataaagcaata



gcatcacaaatttcacaaataaagcatttttttcactgcatt



ctagttgtggtttgtccaaactcatcaatgtatcttatcatg



tctggtactagggttaccccagaacaggtcccattcatggcc



cacatgacaacctgcttccccagtgggtatttttggagacag



ctcttctgtttccaggttttctctcctgcctaaatgtcctgc



ctaagtgccttcaagaacccttcaccatcctgctcctgcatg



tgaccaggttccatggtcagttcaatcacctagtcacagttg



gtaagtgacagagttgggacttgaacctatgcctgcctgaca



ccaagtctttttttgacacctagagccaagacatctgaagac



aaactccctaggagagctggcgtcatagaaaccttaaaggtt



agggagacctgggtttgaatcaggctttgtcagttatgactt



gtgtgaccctagcaagttatttaacctttctgggtctcagtt



tcctcatctgcaaactgaggataataacagtacctaccaaaa



agaactgtcgtgaaaaccatataatttctgcaatgctcctgg



cacagtgtcctgttctaaagcatagttccccttctctttctt



agctccatattgattattaccctaacttgcacaaagagactt



ggaggacccccatagagtatcggagggtcccccatttcctgc



tctttccactccacacccccagcaagcacagggaagttctgg



gggccataatccacccacaggaaccaaatctaagccaccttt



ctggctggtagacatccaggtatgtgggcacagaggtagaca



ggctgaaatgctgctgtgctatcagttgggttttgctggaac



aggaatggaaatggagaggctgacagaactgccctggggagc



ccaggcaagagggacagtggctggacacccccagccagttgt



gcagaccatcagaacaagatcctagattttaggaatacaggg



ttcaagtccgtgcggcaactcttttctaaatatgcccaagcc



attaactttgagttttaaaaatactgatttacaagctgtaca



caatgaaaaaatgcctatccctcacaccatgctgatgctgtt



ccctgccatctcagattaccaattaaatacagaatgcccagt



taaatgtgaactttttttttttttttttttttgagatggagt



tttgttcttgtcgcccaggctagagtgcaatggtgcgatctc



agctcactgcaacctctgcctcccaggttcaagcaattctcc



tgccttagcctcctgagtagctggaactacaggtgcccacca



gcacgcctggctaatttttggtatttttagtggagatggggt



ttcaccatgttggccaggctggtctcgaactcctgacctcag



gtgatctgcctgcctcggcctcccaaagtgctgggattacag



gcgtgagcctaaatgtgaacttttttaatactaaaaaagtat



ttgctgttcatcggaaattcacatttaactgggtgtcctgta



tttttatttgctaaatctaccatcaaattggtctggctcaac



ctggagaatggttaccctaggtaaccacgtgcggaccgagcg



gccgcaggaacccctagtgatggagttggccactccctctct



gcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc



ccgacgcccgggctttgcccgggcggcctcagtgagcgagcg



agcgcgcag










Plasmid TM016 Composition








Δ5′ ITR
 1



occurs at bp 1 through bp 103 of SEQ ID



NO: 31





Human RLBP1
 3


Promoter(short)
occurs at bp 116 through bp 705 of SEQ ID



NO: 31





Modified SV40
 4


intron
occurs at bp 720 through bp 902 of SEQ ID



NO: 31





Added Kozak
 5



occurs at bp 943 through bp 948 of SEQ ID



NO: 31





E_GFP
24



occurs at bp 949 through bp 1668 of SEQ ID



NO: 31





SV40 POLYA
 8



occurs at bp 1726 through bp 1961 of SEQ



ID NO: 31





3′ ITR
 9



occurs at bp 1990 through bp 2119 of SEQ



ID NO: 31





AMP BACTERIAL
15


BACKBONE
occurs at bp 2120 through bp 4738 of SEQ



ID NO: 31





Sequence of
31


TM016 Plasmid
cgcgctcgctcgctcactgaggccgcccgggcaaagcccggg



cgtcgggcgacctttggtcgcccggcctcagtgagcgagcga



gcgcgcagagagggagtggggtaccacgcgtttgtcctctcc



ctgcttggccttaaccagccacatttctcaactgaccccact



cactgcagaggtgaaaactaccatgccaggtcctgctggctg



ggggaggggtgggcaataggcctggatttgccagagctgcca



ctgtagatgtagtcatatttacgatttcccttcacctcttat



taccctggtggtggtggtgggggggggggggtgctctctcag



caaccccaccccgggatcttgaggagaaagagggcagagaaa



agagggaatgggactggcccagatcccagccccacagccggg



cttccacatggccgagcaggaactccagagcaggagcacaca



aaggagggctttgatgcgcctccagccaggcccaggcctctc



ccctctcccctttctctctgggtcttcctttgccccactgag



ggcctcctgtgagcccgatttaacggaaactgtgggcggtga



gaagttccttatgacacactaatcccaacctgctgaccggac



cacgcctccagcggagggaacctctagagctccaggacattc



aggtaccaggtagccccaaggaggagctgccgaatcgatgga



tcgggaactgaaaaaccagaaagttaactggtaagtttagtc



tttttgtcttttatttcaggtcccggatccggtggtggtgca



aatcaaagaactgctcctcagtggatgttgcctttacttcta



ggcctgtacggaagtgttacttctgctctaaaagctgcggaa



ttgtacccgccccgggatccatcgattgaattccccggggat



cctctagagtcgaaattcgccaccatggtgagcaagggcgag



gagctgttcaccggggtggtgcccatcctggtcgagctggac



ggcgacgtaaacggccacaagttcagcgtgtccggcgagggc



gagggcgatgccacctacggcaagctgaccctgaagttcatc



tgcaccaccggcaagctgcccgtgccctggcccaccctcgtg



accaccctgacctacggcgtgcagtgcttcagccgctacccc



gaccacatgaagcagcacgacttcttcaagtccgccatgccc



gaaggctacgtccaggagcgcaccatcttcttcaaggacgac



ggcaactacaagacccgcgccgaggtgaagttcgagggcgac



accctggtgaaccgcatcgagctgaagggcatcgacttcaag



gaggacggcaacatcctggggcacaagctggagtacaactac



aacagccacaacgtctatatcatggccgacaagcagaagaac



ggcatcaaggtgaacttcaagatccgccacaacatcgaggac



ggcagcgtgcagctcgccgaccactaccagcagaacaccccc



atcggcgacggccccgtgctgctgcccgacaaccactacctg



agcacccagtccgccctgagcaaagaccccaacgagaagcgc



gatcacatggtcctgctggagttcgtgaccgccgccgggatc



actctcggcatggacgagctgtacaagtaatagggtaccggt



cgacctgcagaagcttgcctcgagcagcgctgctcgagagat



ctggatcataatcagccataccacatttgtagaggttttact



tgctttaaaaaacctcccacacctccccctgaacctgaaaca



taaaatgaatgcaattgttgttgttaacttgtttattgcagc



ttataatggttacaaataaagcaatagcatcacaaatttcac



aaataaagcatttttttcactgcattctagttgtggtttgtc



caaactcatcaatgtatcttatcatgtctggtaaccacgtgc



ggaccgagcggccgcaggaacccctagtgatggagttggcca



ctccctctctgcgcgctcgctcgctcactgaggccgggcgac



caaaggtcgcccgacgcccgggctttgcccgggcggcctcag



tgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgc



ggtattttctccttacgcatctgtgcggtatttcacaccgca



tacgtcaaagcaaccatagtacgcgccctgtagcggcgcatt



aagcgcggcgggtgtggtggttacgcgcagcgtgaccgctac



acttgccagcgccttagcgcccgctcctttcgctttcttccc



ttcctttctcgccacgttcgccggctttccccgtcaagctct



aaatcgggggctccctttagggttccgatttagtgctttacg



gcacctcgaccccaaaaaacttgatttgggtgatggttcacg



tagtgggccatcgccctgatagacggtttttcgccctttgac



gttggagtccacgttctttaatagtggactcttgttccaaac



tggaacaacactcaactctatctcgggctattcttttgattt



ataagggattttgccgatttcggtctattggttaaaaaatga



gctgatttaacaaaaatttaacgcgaattttaacaaaatatt



aacgtttacaattttatggtgcactctcagtacaatctgctc



tgatgccgcatagttaagccagccccgacacccgccaacacc



cgctgacgcgccctgacgggcttgtctgctcccggcatccgc



ttacagacaagctgtgaccgtctccgggagctgcatgtgtca



gaggttttcaccgtcatcaccgaaacgcgcgagacgaaaggg



cctcgtgatacgcctatttttataggttaatgtcatgataat



aatggtttcttagacgtcaggtggcacttttcggggaaatgt



gcgcggaacccctatttgtttatttttctaaatacattcaaa



tatgtatccgctcatgagacaataaccctgataaatgcttca



ataatattgaaaaaggaagagtatgagtattcaacatttccg



tgtcgcccttattcccttttttgcggcattttgccttcctgt



ttttgctcacccagaaacgctggtgaaagtaaaagatgctga



agatcagttgggtgcacgagtgggttacatcgaactggatct



caacagcggtaagatccttgagagttttcgccccgaagaacg



ttttccaatgatgagcacttttaaagttctgctatgtggcgc



ggtattatcccgtattgacgccgggcaagagcaactcggtcg



ccgcatacactattctcagaatgacttggttgagtactcacc



agtcacagaaaagcatcttacggatggcatgacagtaagaga



attatgcagtgctgccataaccatgagtgataacactgcggc



caacttacttctgacaacgatcggaggaccgaaggagctaac



cgcttttttgcacaacatgggggatcatgtaactcgccttga



tcgttgggaaccggagctgaatgaagccataccaaacgacga



gcgtgacaccacgatgcctgtagcaatggcaacaacgttgcg



caaactattaactggcgaactacttactctagcttcccggca



acaattaatagactggatggaggcggataaagttgcaggacc



acttctgcgctcggcccttccggctggctggtttattgctga



taaatctggagccggtgagcgtgggtctcgcggtatcattgc



agcactggggccagatggtaagccctcccgtatcgtagttat



ctacacgacggggagtcaggcaactatggatgaacgaaatag



acagatcgctgagataggtgcctcactgattaagcattggta



actgtcagaccaagtttactcatatatactttagattgattt



aaaacttcatttttaatttaaaaggatctaggtgaagatcct



ttttgataatctcatgaccaaaatcccttaacgtgagttttc



gttccactgagcgtcagaccccgtagaaaagatcaaaggatc



ttcttgaaatcctttttttctgcgcgtaatctgctgcttgca



aacaaaaaaaccaccgctaccagcggtggtttgtttgccgga



tcaagagctaccaactctttttccgaaggtaactggcttcag



cagagcgcagataccaaatactgttcttctagtgtagccgta



gttaggccaccacttcaagaactctgtagcaccgcctacata



cctcgctctgctaatcctgttaccagtggctgctgccagtgg



cgataagtcgtgtcttaccgggttggactcaagacgatagtt



accggataaggcgcagcggtcgggctgaacggggggttcgtg



cacacagcccagcttggagcgaacgacctacaccgaactgag



atacctacagcgtgagctatgagaaagcgccacgcttcccga



agggagaaaggcggacaggtatccggtaagcggcagggtcgg



aacaggagagcgcacgagggagcttccagggggaaacgcctg



gtatctttatagtcctgtcgggtttcgccacctctgacttga



gcgtcgatttttgtgatgctcgtcaggggggcggagcctatg



gaaaaacgccagcaacgcggcctttttacggttcctggcctt



ttgctggccttttgctcacatgtcctgcaggcagctg





GENE CASSETTE
56


OF PLASMID
cgcgctcgctcgctcactgaggccgcccgggcaaagcccggg


TM016 OCCURS AT
cgtcgggcgacctttggtcgcccggcctcagtgagcgagcga


BP 1 THROUGH
gcgcgcagagagggagtggggtaccacgcgtttgtcctctcc


2119 OF SEQ ID
ctgcttggccttaaccagccacatttctcaactgaccccact


NO: 31
cactgcagaggtgaaaactaccatgccaggtcctgctggctg



ggggaggggtgggcaataggcctggatttgccagagctgcca



ctgtagatgtagtcatatttacgatttcccttcacctcttat



taccctggtggtggtggtgggggggggggggtgctctctcag



caaccccaccccgggatcttgaggagaaagagggcagagaaa



agagggaatgggactggcccagatcccagccccacagccggg



cttccacatggccgagcaggaactccagagcaggagcacaca



aaggagggctttgatgcgcctccagccaggcccaggcctctc



ccctctcccctttctctctgggtcttcctttgccccactgag



ggcctcctgtgagcccgatttaacggaaactgtgggcggtga



gaagttccttatgacacactaatcccaacctgctgaccggac



cacgcctccagcggagggaacctctagagctccaggacattc



aggtaccaggtagccccaaggaggagctgccgaatcgatgga



tcgggaactgaaaaaccagaaagttaactggtaagtttagtc



tttttgtcttttatttcaggtcccggatccggtggtggtgca



aatcaaagaactgctcctcagtggatgttgcctttacttcta



ggcctgtacggaagtgttacttctgctctaaaagctgcggaa



ttgtacccgccccgggatccatcgattgaattccccggggat



cctctagagtcgaaattcgccaccatggtgagcaagggcgag



gagctgttcaccggggtggtgcccatcctggtcgagctggac



ggcgacgtaaacggccacaagttcagcgtgtccggcgagggc



gagggcgatgccacctacggcaagctgaccctgaagttcatc



tgcaccaccggcaagctgcccgtgccctggcccaccctcgtg



accaccctgacctacggcgtgcagtgcttcagccgctacccc



gaccacatgaagcagcacgacttcttcaagtccgccatgccc



gaaggctacgtccaggagcgcaccatcttcttcaaggacgac



ggcaactacaagacccgcgccgaggtgaagttcgagggcgac



accctggtgaaccgcatcgagctgaagggcatcgacttcaag



gaggacggcaacatcctggggcacaagctggagtacaactac



aacagccacaacgtctatatcatggccgacaagcagaagaac



ggcatcaaggtgaacttcaagatccgccacaacatcgaggac



ggcagcgtgcagctcgccgaccactaccagcagaacaccccc



atcggcgacggccccgtgctgctgcccgacaaccactacctg



agcacccagtccgccctgagcaaagaccccaacgagaagcgc



gatcacatggtcctgctggagttcgtgaccgccgccgggatc



actctcggcatggacgagctgtacaagtaatagggtaccggt



cgacctgcagaagcttgcctcgagcagcgctgctcgagagat



ctggatcataatcagccataccacatttgtagaggttttact



tgctttaaaaaacctcccacacctccccctgaacctgaaaca



taaaatgaatgcaattgttgttgttaacttgtttattgcagc



ttataatggttacaaataaagcaatagcatcacaaatttcac



aaataaagcatttttttcactgcattctagttgtggtttgtc



caaactcatcaatgtatcttatcatgtctggtaaccacgtgc



ggaccgagcggccgcaggaacccctagtgatggagttggcca



ctccctctctgcgcgctcgctcgctcactgaggccgggcgac



caaaggtcgcccgacgcccgggctttgcccgggcggcctcag



tgagcgagcgagcgcgcag










Plasmid TM035 Composition








5′ ITR
 2



occurs at bp 1 through bp 119 of SEQ ID



NO: 32





Human RLBP1
10


Promoter(long)
occurs at bp 137 through bp 3293 of SEQ ID



NO: 32





Added Kozak
 5



occurs at bp 3327 through bp 3332 of SEQ



ID NO: 32





E_GFP
24



occurs at bp 3333 through bp 4052 of SEQ



ID NO: 32





SV40 POLYA
 8



occurs at bp 4110 through bp 4345 of SEQ



ID NO: 32





3′ ITR
 9



occurs at bp 4374 through bp 4503 of SEQ



ID NO: 32





AMP BACTERIAL
15


BACKBONE
occurs at bp 4504 through bp 7122 of SEQ



ID NO: 32





Sequence of
32


TM035 Plasmid
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc



gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag



agagggagtggccaactccatcactaggggttcctgcggccg



cacgcagcttttgtcctctccctgcttggccttaaccagcca



catttctcaactgaccccactcactgcagaggtgaaaactac



catgccaggtcctgctggctgggggaggggtgggcaataggc



ctggatttgccagagctgccactgtagatgtagtcatattta



cgatttcccttcacctcttattaccctggtggtggtggtggg



ggggggggggtgctctctcagcaaccccaccccgggatcttg



aggagaaagagggcagagaaaagagggaatgggactggccca



gatcccagccccacagccgggcttccacatggccgagcagga



actccagagcaggagcacacaaaggagggctttgatgcgcct



ccagccaggcccaggcctctcccctctcccctttctctctgg



gtcttcctttgccccactgagggcctcctgtgagcccgattt



aacggaaactgtgggcggtgagaagttccttatgacacacta



atcccaacctgctgaccggaccacgcctccagcggagggaac



ctctagagctccaggacattcaggtaccaggtagccccaagg



aggagctgccgacctggcaggtaagtcaatacctggggcttg



cctgggccagggagcccaggactggggtgaggactcagggga



gcagggagaccacgtcccaagatgcctgtaaaactgaaacca



cctggccattctccaggttgagccagaccaatttgatggcag



atttagcaaataaaaatacaggacacccagttaaatgtgaat



ttcagatgaacagcaaatacttttttagtattaaaaaagttc



acatttaggctcacgcctgtaatcccagcactttgggaggcc



gaggcaggcagatcacctgaggtcaggagttcgagaccagcc



tggccaacatggtgaaaccccatctccactaaaaataccaaa



aattagccaggcgtgctggtgggcacctgtagttccagctac



tcaggaggctaaggcaggagaattgcttgaacctgggaggca



gaggttgcagtgagctgagatcgcaccattgcactctagcct



gggcgacaagaacaaaactccatctcaaaaaaaaaaaaaaaa



aaaaagttcacatttaactgggcattctgtatttaattggta



atctgagatggcagggaacagcatcagcatggtgtgagggat



aggcattttttcattgtgtacagcttgtaaatcagtattttt



aaaactcaaagttaatggcttgggcatatttagaaaagagtt



gccgcacggacttgaaccctgtattcctaaaatctaggatct



tgttctgatggtctgcacaactggctgggggtgtccagccac



tgtccctcttgcctgggctccccagggcagttctgtcagcct



ctccatttccattcctgttccagcaaaacccaactgatagca



cagcagcatttcagcctgtctacctctgtgcccacatacctg



gatgtctaccagccagaaaggtggcttagatttggttcctgt



gggtggattatggcccccagaacttccctgtgcttgctgggg



gtgtggagtggaaagagcaggaaatgggggaccctccgatac



tctatgggggtcctccaagtctctttgtgcaagttagggtaa



taatcaatatggagctaagaaagagaaggggaactatgcttt



agaacaggacactgtgccaggagcattgcagaaattatatgg



ttttcacgacagttctttttggtaggtactgttattatcctc



agtttgcagatgaggaaactgagacccagaaaggttaaataa



cttgctagggtcacacaagtcataactgacaaagcctgattc



aaacccaggtctccctaacctttaaggtttctatgacgccag



ctctcctagggagtttgtcttcagatgtcttggctctaggtg



tcaaaaaaagacttggtgtcaggcaggcataggttcaagtcc



caactctgtcacttaccaactgtgactaggtgattgaactga



ccatggaacctggtcacatgcaggagcaggatggtgaagggt



tcttgaaggcacttaggcaggacatttaggcaggagagaaaa



cctggaaacagaagagctgtctccaaaaatacccactgggga



agcaggttgtcatgtgggccatgaatgggacctgttctggta



accaagcattgcttatgtgtccattacatttcataacacttc



catcctactttacagggaacaaccaagactggggttaaatct



cacagcctgcaagtggaagagaagaacttgaacccaggtcca



acttttgcgccacagcaggctgcctcttggtcctgacaggaa



gtcacaacttgggtctgagtactgatccctggctattttttg



gctgtgttaccttggacaagtcacttattcctcctcccgttt



cctcctatgtaaaatggaaataataatgttgaccctgggtct



gagagagtggatttgaaagtacttagtgcatcacaaagcaca



gaacacacttccagtctcgtgattatgtacttatgtaactgg



tcatcacccatcttgagaatgaatgcattggggaaagggcca



tccactaggctgcgaagtttctgagggactccttcgggctgg



agaaggatggccacaggagggaggagagattgccttatcctg



cagtgatcatgtcattgagaacagagccagattctttttttc



ctggcagggccaacttgttttaacatctaaggactgagctat



ttgtgtctgtgccctttgtccaagcagtgtttcccaaagtgt



agcccaagaaccatctccctcagagccaccaggaagtgcttt



aaattgcaggttcctaggccacagcctgcacctgcagagtca



gaatcatggaggttgggacccaggcacctgcgtttctaacaa



atgcctcgggtgattctgatgcaattgaaagtttgagatcca



cagttctgagacaataacagaatggtttttctaacccctgca



gccctgacttcctatcctagggaaggggccggctggagaggc



caggacagagaaagcagatcccttctttttccaaggactctg



tgtcttccataggcaacgaattccccggggatcctctagagt



cgaaattcgccaccatggtgagcaagggcgaggagctgttca



ccggggtggtgcccatcctggtcgagctggacggcgacgtaa



acggccacaagttcagcgtgtccggcgagggcgagggcgatg



ccacctacggcaagctgaccctgaagttcatctgcaccaccg



gcaagctgcccgtgccctggcccaccctcgtgaccaccctga



cctacggcgtgcagtgcttcagccgctaccccgaccacatga



agcagcacgacttcttcaagtccgccatgcccgaaggctacg



tccaggagcgcaccatcttcttcaaggacgacggcaactaca



agacccgcgccgaggtgaagttcgagggcgacaccctggtga



accgcatcgagctgaagggcatcgacttcaaggaggacggca



acatcctggggcacaagctggagtacaactacaacagccaca



acgtctatatcatggccgacaagcagaagaacggcatcaagg



tgaacttcaagatccgccacaacatcgaggacggcagcgtgc



agctcgccgaccactaccagcagaacacccccatcggcgacg



gccccgtgctgctgcccgacaaccactacctgagcacccagt



ccgccctgagcaaagaccccaacgagaagcgcgatcacatgg



tcctgctggagttcgtgaccgccgccgggatcactctcggca



tggacgagctgtacaagtaatagggtaccggtcgacctgcag



aagcttgcctcgagcagcgctgctcgagagatctggatcata



atcagccataccacatttgtagaggttttacttgctttaaaa



aacctcccacacctccccctgaacctgaaacataaaatgaat



gcaattgttgttgttaacttgtttattgcagcttataatggt



tacaaataaagcaatagcatcacaaatttcacaaataaagca



tttttttcactgcattctagttgtggtttgtccaaactcatc



aatgtatcttatcatgtctggtaaccacgtgcggaccgagcg



gccgcaggaacccctagtgatggagttggccactccctctct



gcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc



ccgacgcccgggctttgcccgggcggcctcagtgagcgagcg



agcgcgcagctgcctgcaggggcgcctgatgcggtattttct



ccttacgcatctgtgcggtatttcacaccgcatacgtcaaag



caaccatagtacgcgccctgtagcggcgcattaagcgcggcg



ggtgtggtggttacgcgcagcgtgaccgctacacttgccagc



gccttagcgcccgctcctttcgctttcttcccttcctttctc



gccacgttcgccggctttccccgtcaagctctaaatcggggg



ctccctttagggttccgatttagtgctttacggcacctcgac



cccaaaaaacttgatttgggtgatggttcacgtagtgggcca



tcgccctgatagacggtttttcgccctttgacgttggagtcc



acgttctttaatagtggactcttgttccaaactggaacaaca



ctcaactctatctcgggctattcttttgatttataagggatt



ttgccgatttcggtctattggttaaaaaatgagctgatttaa



caaaaatttaacgcgaattttaacaaaatattaacgtttaca



attttatggtgcactctcagtacaatctgctctgatgccgca



tagttaagccagccccgacacccgccaacacccgctgacgcg



ccctgacgggcttgtctgctcccggcatccgcttacagacaa



gctgtgaccgtctccgggagctgcatgtgtcagaggttttca



ccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgata



cgcctatttttataggttaatgtcatgataataatggtttct



tagacgtcaggtggcacttttcggggaaatgtgcgcggaacc



cctatttgtttatttttctaaatacattcaaatatgtatccg



ctcatgagacaataaccctgataaatgcttcaataatattga



aaaaggaagagtatgagtattcaacatttccgtgtcgccctt



attcccttttttgcggcattttgccttcctgtttttgctcac



ccagaaacgctggtgaaagtaaaagatgctgaagatcagttg



ggtgcacgagtgggttacatcgaactggatctcaacagcggt



aagatccttgagagttttcgccccgaagaacgttttccaatg



atgagcacttttaaagttctgctatgtggcgcggtattatcc



cgtattgacgccgggcaagagcaactcggtcgccgcatacac



tattctcagaatgacttggttgagtactcaccagtcacagaa



aagcatcttacggatggcatgacagtaagagaattatgcagt



gctgccataaccatgagtgataacactgcggccaacttactt



ctgacaacgatcggaggaccgaaggagctaaccgcttttttg



cacaacatgggggatcatgtaactcgccttgatcgttgggaa



ccggagctgaatgaagccataccaaacgacgagcgtgacacc



acgatgcctgtagcaatggcaacaacgttgcgcaaactatta



actggcgaactacttactctagcttcccggcaacaattaata



gactggatggaggcggataaagttgcaggaccacttctgcgc



tcggcccttccggctggctggtttattgctgataaatctgga



gccggtgagcgtgggtctcgcggtatcattgcagcactgggg



ccagatggtaagccctcccgtatcgtagttatctacacgacg



gggagtcaggcaactatggatgaacgaaatagacagatcgct



gagataggtgcctcactgattaagcattggtaactgtcagac



caagtttactcatatatactttagattgatttaaaacttcat



ttttaatttaaaaggatctaggtgaagatcctttttgataat



ctcatgaccaaaatcccttaacgtgagttttcgttccactga



gcgtcagaccccgtagaaaagatcaaaggatcttcttgaaat



cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaa



ccaccgctaccagcggtggtttgtttgccggatcaagagcta



ccaactctttttccgaaggtaactggcttcagcagagcgcag



ataccaaatactgttcttctagtgtagccgtagttaggccac



cacttcaagaactctgtagcaccgcctacatacctcgctctg



ctaatcctgttaccagtggctgctgccagtggcgataagtcg



tgtcttaccgggttggactcaagacgatagttaccggataag



gcgcagcggtcgggctgaacggggggttcgtgcacacagccc



agcttggagcgaacgacctacaccgaactgagatacctacag



cgtgagctatgagaaagcgccacgcttcccgaagggagaaag



gcggacaggtatccggtaagcggcagggtcggaacaggagag



cgcacgagggagcttccagggggaaacgcctggtatctttat



agtcctgtcgggtttcgccacctctgacttgagcgtcgattt



ttgtgatgctcgtcaggggggcggagcctatggaaaaacgcc



agcaacgcggcctttttacggttcctggccttttgctggcct



tttgctcacatgtcctgcaggcag





GENE CASSETTE
57


OF PLASMID
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


TM035 OCCURS AT
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


BP 1 THROUGH
agagggagtggccaactccatcactaggggttcctgcggccg


4503 OF SEQ ID
cacgcagcttttgtcctctccctgcttggccttaaccagcca


NO: 32
catttctcaactgaccccactcactgcagaggtgaaaactac



catgccaggtcctgctggctgggggaggggtgggcaataggc



ctggatttgccagagctgccactgtagatgtagtcatattta



cgatttcccttcacctcttattaccctggtggtggtggtggg



ggggggggggtgctctctcagcaaccccaccccgggatcttg



aggagaaagagggcagagaaaagagggaatgggactggccca



gatcccagccccacagccgggcttccacatggccgagcagga



actccagagcaggagcacacaaaggagggctttgatgcgcct



ccagccaggcccaggcctctcccctctcccctttctctctgg



gtcttcctttgccccactgagggcctcctgtgagcccgattt



aacggaaactgtgggcggtgagaagttccttatgacacacta



atcccaacctgctgaccggaccacgcctccagcggagggaac



ctctagagctccaggacattcaggtaccaggtagccccaagg



aggagctgccgacctggcaggtaagtcaatacctggggcttg



cctgggccagggagcccaggactggggtgaggactcagggga



gcagggagaccacgtcccaagatgcctgtaaaactgaaacca



cctggccattctccaggttgagccagaccaatttgatggcag



atttagcaaataaaaatacaggacacccagttaaatgtgaat



ttcagatgaacagcaaatacttttttagtattaaaaaagttc



acatttaggctcacgcctgtaatcccagcactttgggaggcc



gaggcaggcagatcacctgaggtcaggagttcgagaccagcc



tggccaacatggtgaaaccccatctccactaaaaataccaaa



aattagccaggcgtgctggtgggcacctgtagttccagctac



tcaggaggctaaggcaggagaattgcttgaacctgggaggca



gaggttgcagtgagctgagatcgcaccattgcactctagcct



gggcgacaagaacaaaactccatctcaaaaaaaaaaaaaaaa



aaaaagttcacatttaactgggcattctgtatttaattggta



atctgagatggcagggaacagcatcagcatggtgtgagggat



aggcattttttcattgtgtacagcttgtaaatcagtattttt



aaaactcaaagttaatggcttgggcatatttagaaaagagtt



gccgcacggacttgaaccctgtattcctaaaatctaggatct



tgttctgatggtctgcacaactggctgggggtgtccagccac



tgtccctcttgcctgggctccccagggcagttctgtcagcct



ctccatttccattcctgttccagcaaaacccaactgatagca



cagcagcatttcagcctgtctacctctgtgcccacatacctg



gatgtctaccagccagaaaggtggcttagatttggttcctgt



gggtggattatggcccccagaacttccctgtgcttgctgggg



gtgtggagtggaaagagcaggaaatgggggaccctccgatac



tctatgggggtcctccaagtctctttgtgcaagttagggtaa



taatcaatatggagctaagaaagagaaggggaactatgcttt



agaacaggacactgtgccaggagcattgcagaaattatatgg



ttttcacgacagttctttttggtaggtactgttattatcctc



agtttgcagatgaggaaactgagacccagaaaggttaaataa



cttgctagggtcacacaagtcataactgacaaagcctgattc



aaacccaggtctccctaacctttaaggtttctatgacgccag



ctctcctagggagtttgtcttcagatgtcttggctctaggtg



tcaaaaaaagacttggtgtcaggcaggcataggttcaagtcc



caactctgtcacttaccaactgtgactaggtgattgaactga



ccatggaacctggtcacatgcaggagcaggatggtgaagggt



tcttgaaggcacttaggcaggacatttaggcaggagagaaaa



cctggaaacagaagagctgtctccaaaaatacccactgggga



agcaggttgtcatgtgggccatgaatgggacctgttctggta



accaagcattgcttatgtgtccattacatttcataacacttc



catcctactttacagggaacaaccaagactggggttaaatct



cacagcctgcaagtggaagagaagaacttgaacccaggtcca



acttttgcgccacagcaggctgcctcttggtcctgacaggaa



gtcacaacttgggtctgagtactgatccctggctattttttg



gctgtgttaccttggacaagtcacttattcctcctcccgttt



cctcctatgtaaaatggaaataataatgttgaccctgggtct



gagagagtggatttgaaagtacttagtgcatcacaaagcaca



gaacacacttccagtctcgtgattatgtacttatgtaactgg



tcatcacccatcttgagaatgaatgcattggggaaagggcca



tccactaggctgcgaagtttctgagggactccttcgggctgg



agaaggatggccacaggagggaggagagattgccttatcctg



cagtgatcatgtcattgagaacagagccagattctttttttc



ctggcagggccaacttgttttaacatctaaggactgagctat



ttgtgtctgtgccctttgtccaagcagtgtttcccaaagtgt



agcccaagaaccatctccctcagagccaccaggaagtgcttt



aaattgcaggttcctaggccacagcctgcacctgcagagtca



gaatcatggaggttgggacccaggcacctgcgtttctaacaa



atgcctcgggtgattctgatgcaattgaaagtttgagatcca



cagttctgagacaataacagaatggtttttctaacccctgca



gccctgacttcctatcctagggaaggggccggctggagaggc



caggacagagaaagcagatcccttctttttccaaggactctg



tgtcttccataggcaacgaattccccggggatcctctagagt



cgaaattcgccaccatggtgagcaagggcgaggagctgttca



ccggggtggtgcccatcctggtcgagctggacggcgacgtaa



acggccacaagttcagcgtgtccggcgagggcgagggcgatg



ccacctacggcaagctgaccctgaagttcatctgcaccaccg



gcaagctgcccgtgccctggcccaccctcgtgaccaccctga



cctacggcgtgcagtgcttcagccgctaccccgaccacatga



agcagcacgacttcttcaagtccgccatgcccgaaggctacg



tccaggagcgcaccatcttcttcaaggacgacggcaactaca



agacccgcgccgaggtgaagttcgagggcgacaccctggtga



accgcatcgagctgaagggcatcgacttcaaggaggacggca



acatcctggggcacaagctggagtacaactacaacagccaca



acgtctatatcatggccgacaagcagaagaacggcatcaagg



tgaacttcaagatccgccacaacatcgaggacggcagcgtgc



agctcgccgaccactaccagcagaacacccccatcggcgacg



gccccgtgctgctgcccgacaaccactacctgagcacccagt



ccgccctgagcaaagaccccaacgagaagcgcgatcacatgg



tcctgctggagttcgtgaccgccgccgggatcactctcggca



tggacgagctgtacaagtaatagggtaccggtcgacctgcag



aagcttgcctcgagcagcgctgctcgagagatctggatcata



atcagccataccacatttgtagaggttttacttgctttaaaa



aacctcccacacctccccctgaacctgaaacataaaatgaat



gcaattgttgttgttaacttgtttattgcagcttataatggt



tacaaataaagcaatagcatcacaaatttcacaaataaagca



tttttttcactgcattctagttgtggtttgtccaaactcatc



aatgtatcttatcatgtctggtaaccacgtgcggaccgagcg



gccgcaggaacccctagtgatggagttggccactccctctct



gcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc



ccgacgcccgggctttgcccgggcggcctcagtgagcgagcg



agcgcgcag










Plasmid AG012 Composition








5′ ITR
 2



occurs @ bp 1 through bp 119 of SEQ ID NO:



33





SYNUCLEIN
13


INTRONIC
occurs @ bp 148 through bp 2601 of SEQ ID


SEQUENCE AS
NO: 33


STUFFER


SEQUENCE





SV40 POLYA
 8



occurs @ bp 2640 through bp 2875 of SEQ ID



NO: 33





RLBP1 INTRONIC
14


SEQUENCE AS
occurs at bp 2883 through bp 4385 of SEQ


STUFFER
ID NO: 33


SEQUENCE





3′ ITR
 9



occurs at bp 4414 through bp 4543 of SEQ



ID NO: 33





AMP BACTERIAL
15


BACKBONE
occurs at bp 4544 through bp 7162 of SEQ



ID NO: 33





Sequence of
33


AG012 Plasmid
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc



gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag



agagggagtggccaactccatcactaggggttcctgcggccg



cacgcgtgacgtcgtttaaacgggccccggtgttatctcatt



cttttttctcctctgtaagttgacatgtgatgtgggaacaaa



ggggataaagtcattattttgtgctaaaatcgtaattggaga



ggacctcctgttagctgggctttcttctatttattgtggtgg



ttactggagttccttcttctagttttaggatatatatatata



ttttttttttttctttccctgaagatataataatatatatac



ttctgaagattgagatttttaaattagttgtattgaaaacta



gctaatcagcaatttaaggctagcttgagacttatgtcttga



atttgtttttgtaggctccaaaaccaaggagggagtggtgca



tggtgtggcaacaggtaagctccattgtgcttatatccaaag



atgatatttaaagtatctagtgattagtgtggcccagtattc



aagattcctatgaaattgtaaaacaatcactgagcattctaa



gaacatatcagtcttattgaaactgaattctttataaagtat



ttttaaaaaggtaaatattgattataaataaaaaatatactt



gccaagaataatgagggctttgaattgataagctatgtttaa



tttatagtaagtgggcatttaaatattctgaccaaaaatgta



ttgacaaactgctgacaaaaataaaatgtgaatattgccata



attttaaaaaaagagtaaaatttctgttgattacagtaaaat



attttgaccttaaattatgttgattacaatattcctttgata



attcagagtgcatttcaggaaacacccttggacagtcagtaa



attgtttattgtatttatctttgtattgttatggtatagcta



tttgtacaaatattattgtgcaattattacatttctgattat



attattcatttggcctaaatttaccaagaatttgaacaagtc



aattaggtttacaatcaagaaatatcaaaaatgatgaaaagg



atgataatcatcatcagatgttgaggaagatgacgatgagag



tgccagaaatagagaaatcaaaggagaaccaaaatttaacaa



attaaaagcccacagacttgctgtaattaagttttctgttgt



aagtactccacgtttcctggcagatgtggtgaagcaaaagat



ataatcagaaatataatttatatgatcggaaagcattaaaca



caatagtgcctatacaaataaaatgttcctatcactgacttc



taaaatggaaatgaggacaatgatatgggaatcttaatacag



tgttgtggataggactaaaaacacaggagtcagatcttcttg



gttcaacttcctgcttactccttaccagctgtgtgttttttg



caaggttcttcacctctatgtgatttagcttcctcatctata



aaataattcagtgaattaatgtacacaaaacatctggaaaac



aaaagcaaacaatatgtattttataagtgttacttatagttt



tatagtgaactttcttgtgcaacatttttacaactagtggag



aaaaatatttctttaaatgaatacttttgatttaaaaatcag



agtgtaaaaataaaacagactcctttgaaactagttctgtta



gaagttaattgtgcacctttaatgggctctgttgcaatccaa



cagagaagtagttaagtaagtggactatgatggcttctaggg



acctcctataaatatgatattgtgaagcatgattataataag



aactagataacagacaggtggagactccactatctgaagagg



gtcaacctagatgaatggtgttccatttagtagttgaggaag



aacccatgaggtttagaaagcagacaagcatgtggcaagttc



tggagtcagtggtaaaaattaaagaacccaactattactgtc



acctaatgatctaatggagactgtggagatgggctgcatttt



tttaatcttctccagaatgccaaaatgtaaacacatatctgt



gtgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagagaga



gagagagagagactgaagtttgtacaattagacattttataa



aatgttttctgaaggacagtggctcacaatcttaagtttcta



acattgtacaatgttgggagactttgtatactttattttctc



tttagcatattaaggaatctgagatgtcctacagtaaagaaa



tttgcattacatagttaaaatcagggttattcaaactttttg



attattgaaacctttcttcattagttactagggttgaatgaa



actagtgttccacagaaaactatgggaaatgttgctaggcag



taaggacatggtgatttcagcatgtgcaatatttacagcgat



tgcacccatggaccaccctggcagtagtgaaataaccaaaaa



tgctgtcataactagtatggctatgagaaacacattgggcag



aagcttgcctcgagcagcgctgctcgagagatctggatcata



atcagccataccacatttgtagaggttttacttgctttaaaa



aacctcccacacctccccctgaacctgaaacataaaatgaat



gcaattgttgttgttaacttgtttattgcagcttataatggt



tacaaataaagcaatagcatcacaaatttcacaaataaagca



tttttttcactgcattctagttgtggtttgtccaaactcatc



aatgtatcttatcatgtctggtaaccattctccaggttgagc



cagaccaatttgatggtagatttagcaaataaaaatacagga



cacccagttaaatgtgaatttccgatgaacagcaaatacttt



tttagtattaaaaaagttcacatttaggctcacgcctgtaat



cccagcactttgggaggccgaggcaggcagatcacctgaggt



caggagttcgagaccagcctggccaacatggtgaaaccccat



ctccactaaaaataccaaaaattagccaggcgtgctggtggg



cacctgtagttccagctactcaggaggctaaggcaggagaat



tgcttgaacctgggaggcagaggttgcagtgagctgagatcg



caccattgcactctagcctgggcgacaagaacaaaactccat



ctcaaaaaaaaaaaaaaaaaaaaagttcacatttaactgggc



attctgtatttaattggtaatctgagatggcagggaacagca



tcagcatggtgtgagggataggcattttttcattgtgtacag



cttgtaaatcagtatttttaaaactcaaagttaatggcttgg



gcatatttagaaaagagttgccgcacggacttgaaccctgta



ttcctaaaatctaggatcttgttctgatggtctgcacaactg



gctgggggtgtccagccactgtccctcttgcctgggctcccc



agggcagttctgtcagcctctccatttccattcctgttccag



caaaacccaactgatagcacagcagcatttcagcctgtctac



ctctgtgcccacatacctggatgtctaccagccagaaaggtg



gcttagatttggttcctgtgggtggattatggcccccagaac



ttccctgtgcttgctgggggtgtggagtggaaagagcaggaa



atgggggaccctccgatactctatgggggtcctccaagtctc



tttgtgcaagttagggtaataatcaatatggagctaagaaag



agaaggggaactatgctttagaacaggacactgtgccaggag



cattgcagaaattatatggttttcacgacagttctttttggt



aggtactgttattatcctcagtttgcagatgaggaaactgag



acccagaaaggttaaataacttgctagggtcacacaagtcat



aactgacaaagcctgattcaaacccaggtctccctaaccttt



aaggtttctatgacgccagctctcctagggagtttgtcttca



gatgtcttggctctaggtgtcaaaaaaagacttggtgtcagg



caggcataggttcaagtcccaactctgtcacttaccaactgt



gactaggtgattgaactgaccatggaacctggtcacatgcag



gagcaggatggtgaagggttcttgaaggcacttaggcaggac



atttaggcaggagagaaaacctggaaacagaagagctgtctc



caaaaatacccactggggaagcaggttgtcatgtgggccatg



aatgggacctgttctggggtaaccacgtgcggaccgagcggc



cgcaggaacccctagtgatggagttggccactccctctctgc



gcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc



gacgcccgggctttgcccgggcggcctcagtgagcgagcgag



cgcgcagctgcctgcaggggcgcctgatgcggtattttctcc



ttacgcatctgtgcggtatttcacaccgcatacgtcaaagca



accatagtacgcgccctgtagcggcgcattaagcgcggcggg



tgtggtggttacgcgcagcgtgaccgctacacttgccagcgc



cttagcgcccgctcctttcgctttcttcccttcctttctcgc



cacgttcgccggctttccccgtcaagctctaaatcgggggct



ccctttagggttccgatttagtgctttacggcacctcgaccc



caaaaaacttgatttgggtgatggttcacgtagtgggccatc



gccctgatagacggtttttcgccctttgacgttggagtccac



gttctttaatagtggactcttgttccaaactggaacaacact



caactctatctcgggctattcttttgatttataagggatttt



gccgatttcggtctattggttaaaaaatgagctgatttaaca



aaaatttaacgcgaattttaacaaaatattaacgtttacaat



tttatggtgcactctcagtacaatctgctctgatgccgcata



gttaagccagccccgacacccgccaacacccgctgacgcgcc



ctgacgggcttgtctgctcccggcatccgcttacagacaagc



tgtgaccgtctccgggagctgcatgtgtcagaggttttcacc



gtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacg



cctatttttataggttaatgtcatgataataatggtttctta



gacgtcaggtggcacttttcggggaaatgtgcgcggaacccc



tatttgtttatttttctaaatacattcaaatatgtatccgct



catgagacaataaccctgataaatgcttcaataatattgaaa



aaggaagagtatgagtattcaacatttccgtgtcgcccttat



tcccttttttgcggcattttgccttcctgtttttgctcaccc



agaaacgctggtgaaagtaaaagatgctgaagatcagttggg



tgcacgagtgggttacatcgaactggatctcaacagcggtaa



gatccttgagagttttcgccccgaagaacgttttccaatgat



gagcacttttaaagttctgctatgtggcgcggtattatcccg



tattgacgccgggcaagagcaactcggtcgccgcatacacta



ttctcagaatgacttggttgagtactcaccagtcacagaaaa



gcatcttacggatggcatgacagtaagagaattatgcagtgc



tgccataaccatgagtgataacactgcggccaacttacttct



gacaacgatcggaggaccgaaggagctaaccgcttttttgca



caacatgggggatcatgtaactcgccttgatcgttgggaacc



ggagctgaatgaagccataccaaacgacgagcgtgacaccac



gatgcctgtagcaatggcaacaacgttgcgcaaactattaac



tggcgaactacttactctagcttcccggcaacaattaataga



ctggatggaggcggataaagttgcaggaccacttctgcgctc



ggcccttccggctggctggtttattgctgataaatctggagc



cggtgagcgtgggtctcgcggtatcattgcagcactggggcc



agatggtaagccctcccgtatcgtagttatctacacgacggg



gagtcaggcaactatggatgaacgaaatagacagatcgctga



gataggtgcctcactgattaagcattggtaactgtcagacca



agtttactcatatatactttagattgatttaaaacttcattt



ttaatttaaaaggatctaggtgaagatcctttttgataatct



catgaccaaaatcccttaacgtgagttttcgttccactgagc



gtcagaccccgtagaaaagatcaaaggatcttcttgaaatcc



tttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacc



accgctaccagcggtggtttgtttgccggatcaagagctacc



aactctttttccgaaggtaactggcttcagcagagcgcagat



accaaatactgttcttctagtgtagccgtagttaggccacca



cttcaagaactctgtagcaccgcctacatacctcgctctgct



aatcctgttaccagtggctgctgccagtggcgataagtcgtg



tcttaccgggttggactcaagacgatagttaccggataaggc



gcagcggtcgggctgaacggggggttcgtgcacacagcccag



cttggagcgaacgacctacaccgaactgagatacctacagcg



tgagctatgagaaagcgccacgcttcccgaagggagaaaggc



ggacaggtatccggtaagcggcagggtcggaacaggagagcg



cacgagggagcttccagggggaaacgcctggtatctttatag



tcctgtcgggtttcgccacctctgacttgagcgtcgattttt



gtgatgctcgtcaggggggcggagcctatggaaaaacgccag



caacgcggcctttttacggttcctggccttttgctggccttt



tgctcacatgtcctgcaggcag





INSERT OF
58


PLASMID AG012
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


OCCURS AT BP 1
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


THROUGH 4543 OF
agagggagtggccaactccatcactaggggttcctgcggccg


SEQ ID NO: 33
cacgcgtgacgtcgtttaaacgggccccggtgttatctcatt


(USED AS
cttttttctcctctgtaagttgacatgtgatgtgggaacaaa


NEGATIVE
ggggataaagtcattattttgtgctaaaatcgtaattggaga


CONTROL FOR
ggacctcctgttagctgggctttcttctatttattgtggtgg


GENE CASSETTE)
ttactggagttccttcttctagttttaggatatatatatata



ttttttttttttctttccctgaagatataataatatatatac



ttctgaagattgagatttttaaattagttgtattgaaaacta



gctaatcagcaatttaaggctagcttgagacttatgtcttga



atttgtttttgtaggctccaaaaccaaggagggagtggtgca



tggtgtggcaacaggtaagctccattgtgcttatatccaaag



atgatatttaaagtatctagtgattagtgtggcccagtattc



aagattcctatgaaattgtaaaacaatcactgagcattctaa



gaacatatcagtcttattgaaactgaattctttataaagtat



ttttaaaaaggtaaatattgattataaataaaaaatatactt



gccaagaataatgagggctttgaattgataagctatgtttaa



tttatagtaagtgggcatttaaatattctgaccaaaaatgta



ttgacaaactgctgacaaaaataaaatgtgaatattgccata



attttaaaaaaagagtaaaatttctgttgattacagtaaaat



attttgaccttaaattatgttgattacaatattcctttgata



attcagagtgcatttcaggaaacacccttggacagtcagtaa



attgtttattgtatttatctttgtattgttatggtatagcta



tttgtacaaatattattgtgcaattattacatttctgattat



attattcatttggcctaaatttaccaagaatttgaacaagtc



aattaggtttacaatcaagaaatatcaaaaatgatgaaaagg



atgataatcatcatcagatgttgaggaagatgacgatgagag



tgccagaaatagagaaatcaaaggagaaccaaaatttaacaa



attaaaagcccacagacttgctgtaattaagttttctgttgt



aagtactccacgtttcctggcagatgtggtgaagcaaaagat



ataatcagaaatataatttatatgatcggaaagcattaaaca



caatagtgcctatacaaataaaatgttcctatcactgacttc



taaaatggaaatgaggacaatgatatgggaatcttaatacag



tgttgtggataggactaaaaacacaggagtcagatcttcttg



gttcaacttcctgcttactccttaccagctgtgtgttttttg



caaggttcttcacctctatgtgatttagcttcctcatctata



aaataattcagtgaattaatgtacacaaaacatctggaaaac



aaaagcaaacaatatgtattttataagtgttacttatagttt



tatagtgaactttcttgtgcaacatttttacaactagtggag



aaaaatatttctttaaatgaatacttttgatttaaaaatcag



agtgtaaaaataaaacagactcctttgaaactagttctgtta



gaagttaattgtgcacctttaatgggctctgttgcaatccaa



cagagaagtagttaagtaagtggactatgatggcttctaggg



acctcctataaatatgatattgtgaagcatgattataataag



aactagataacagacaggtggagactccactatctgaagagg



gtcaacctagatgaatggtgttccatttagtagttgaggaag



aacccatgaggtttagaaagcagacaagcatgtggcaagttc



tggagtcagtggtaaaaattaaagaacccaactattactgtc



acctaatgatctaatggagactgtggagatgggctgcatttt



tttaatcttctccagaatgccaaaatgtaaacacatatctgt



gtgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagagaga



gagagagagagactgaagtttgtacaattagacattttataa



aatgttttctgaaggacagtggctcacaatcttaagtttcta



acattgtacaatgttgggagactttgtatactttattttctc



tttagcatattaaggaatctgagatgtcctacagtaaagaaa



tttgcattacatagttaaaatcagggttattcaaactttttg



attattgaaacctttcttcattagttactagggttgaatgaa



actagtgttccacagaaaactatgggaaatgttgctaggcag



taaggacatggtgatttcagcatgtgcaatatttacagcgat



tgcacccatggaccaccctggcagtagtgaaataaccaaaaa



tgctgtcataactagtatggctatgagaaacacattgggcag



aagcttgcctcgagcagcgctgctcgagagatctggatcata



atcagccataccacatttgtagaggttttacttgctttaaaa



aacctcccacacctccccctgaacctgaaacataaaatgaat



gcaattgttgttgttaacttgtttattgcagcttataatggt



tacaaataaagcaatagcatcacaaatttcacaaataaagca



tttttttcactgcattctagttgtggtttgtccaaactcatc



aatgtatcttatcatgtctggtaaccattctccaggttgagc



cagaccaatttgatggtagatttagcaaataaaaatacagga



cacccagttaaatgtgaatttccgatgaacagcaaatacttt



tttagtattaaaaaagttcacatttaggctcacgcctgtaat



cccagcactttgggaggccgaggcaggcagatcacctgaggt



caggagttcgagaccagcctggccaacatggtgaaaccccat



ctccactaaaaataccaaaaattagccaggcgtgctggtggg



cacctgtagttccagctactcaggaggctaaggcaggagaat



tgcttgaacctgggaggcagaggttgcagtgagctgagatcg



caccattgcactctagcctgggcgacaagaacaaaactccat



ctcaaaaaaaaaaaaaaaaaaaaagttcacatttaactgggc



attctgtatttaattggtaatctgagatggcagggaacagca



tcagcatggtgtgagggataggcattttttcattgtgtacag



cttgtaaatcagtatttttaaaactcaaagttaatggcttgg



gcatatttagaaaagagttgccgcacggacttgaaccctgta



ttcctaaaatctaggatcttgttctgatggtctgcacaactg



gctgggggtgtccagccactgtccctcttgcctgggctcccc



agggcagttctgtcagcctctccatttccattcctgttccag



caaaacccaactgatagcacagcagcatttcagcctgtctac



ctctgtgcccacatacctggatgtctaccagccagaaaggtg



gcttagatttggttcctgtgggtggattatggcccccagaac



ttccctgtgcttgctgggggtgtggagtggaaagagcaggaa



atgggggaccctccgatactctatgggggtcctccaagtctc



tttgtgcaagttagggtaataatcaatatggagctaagaaag



agaaggggaactatgctttagaacaggacactgtgccaggag



cattgcagaaattatatggttttcacgacagttctttttggt



aggtactgttattatcctcagtttgcagatgaggaaactgag



acccagaaaggttaaataacttgctagggtcacacaagtcat



aactgacaaagcctgattcaaacccaggtctccctaaccttt



aaggtttctatgacgccagctctcctagggagtttgtcttca



gatgtcttggctctaggtgtcaaaaaaagacttggtgtcagg



caggcataggttcaagtcccaactctgtcacttaccaactgt



gactaggtgattgaactgaccatggaacctggtcacatgcag



gagcaggatggtgaagggttcttgaaggcacttaggcaggac



atttaggcaggagagaaaacctggaaacagaagagctgtctc



caaaaatacccactggggaagcaggttgtcatgtgggccatg



aatgggacctgttctggggtaaccacgtgcggaccgagcggc



cgcaggaacccctagtgatggagttggccactccctctctgc



gcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc



gacgcccgggctttgcccgggcggcctcagtgagcgagcgag



cgcgcag










Plasmid AG004 Composition








5′ ITR
 2



occurs @ bp 1 through bp 119 of SEQ ID



NO: 34





Human RPE65
11


Promoter
occurs @ bp 134 through bp 1718 of SEQ ID



NO: 34





Added Kozak
 5



occurs @ bp 1752 through 1757 of of SEQ ID



NO: 34





E-GFP
24



occurs @ bp 1758 through bp 2477 of SEQ



ID NO: 34





SV40 POLYA
 8



occurs at bp 2535 through bp 2770 of SEQ



ID NO: 34





RLBP1 INTRONIC
14


SEQUENCE AS
occurs at bp 2778 through bp 4280 of SEQ


STUFFER
ID NO: 34


SEQUENCE





3′ ITR
 9



occurs at bp 4309 through bp 4438 of SEQ



ID NO: 34





AMP BACTERIAL
15


BACKBONE
occurs at bp 4439 through bp 7057 of SEQ



ID NO: 34





Sequence of
34


plasmid AG004
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc



gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag



agagggagtggccaactccatcactaggggttcctgcggccg



cacgcgttacgtaatatttattgaagtttaatattgtgtttg



tgatacagaagtatttgctttaattctaaataaaaattttat



gcttttattgctggtttaagaagatttggattatccttgtac



tttgaggagaagtttcttatttgaaatattttggaaacaggt



cttttaatgtggaaagatagatattaatctcctcttctatta



ctctccaagatccaacaaaagtgattataccccccaaaatat



gatggtagtatcttatactaccatcattttataggcataggg



ctcttagctgcaaataatggaactaactctaataaagcagaa



cgcaaatattgtaaatattagagagctaacaatctctgggat



ggctaaaggatggagcttggaggctacccagccagtaacaat



attccgggctccactgttgaatggagacactacaactgcctt



ggatgggcagagatattatggatgctaagccccaggtgctac



cattaggacttctaccactgtccctaacgggtggagcccatc



acatgcctatgccctcactgtaaggaaatgaagctactgttg



tatatcttgggaagcacttggattaattgttatacagttttg



ttgaagaagacccctagggtaagtagccataactgcacacta



aatttaaaattgttaatgagtttctcaaaaaaaatgttaagg



ttgttagctggtatagtatatatcttgcctgttttccaagga



cttctttgggcagtaccttgtctgtgctggcaagcaactgag



acttaatgaaagagtattggagatatgaatgaattgatgctg



tatactctcagagtgccaaacatataccaatggacaagaagg



tgaggcagagagcagacaggcattagtgacaagcaaagatat



gcagaatttcattctcagcaaatcaaaagtcctcaacctggt



tggaagaatattggcactgaatggtatcaataaggttgctag



agagggttagaggtgcacaatgtgcttccataacattttata



cttctccaatcttagcactaatcaaacatggttgaatacttt



gtttactataactcttacagagttataagatctgtgaagaca



gggacagggacaatacccatctctgtctggttcataggtggt



atgtaatagatatttttaaaaataagtgagttaatgaatgag



ggtgagaatgaaggcacagaggtattagggggaggtgggccc



cagagaatggtgccaaggtccagtggggtgactgggatcagc



tcaggcctgacgctggccactcccacctagctcctttctttc



taatctgttctcattctccttgggaaggattgaggtctctgg



aaaacagccaaacaactgttatgggaacagcaagcccaaata



aagccaagcatcagggggatctgagagctgaaagcaacttct



gttccccctccctcagctgaaggggtggggaagggctcccaa



agccataactccttttaagggatttagaaggcataaaaaggc



ccctggctgagaacttccttcttcattctgcagttggtgaat



tccccggggatcctctagagtcgaaattcgccaccatggtga



gcaagggcgaggagctgttcaccggggtggtgcccatcctgg



tcgagctggacggcgacgtaaacggccacaagttcagcgtgt



ccggcgagggcgagggcgatgccacctacggcaagctgaccc



tgaagttcatctgcaccaccggcaagctgcccgtgccctggc



ccaccctcgtgaccaccctgacctacggcgtgcagtgcttca



gccgctaccccgaccacatgaagcagcacgacttcttcaagt



ccgccatgcccgaaggctacgtccaggagcgcaccatcttct



tcaaggacgacggcaactacaagacccgcgccgaggtgaagt



tcgagggcgacaccctggtgaaccgcatcgagctgaagggca



tcgacttcaaggaggacggcaacatcctggggcacaagctgg



agtacaactacaacagccacaacgtctatatcatggccgaca



agcagaagaacggcatcaaggtgaacttcaagatccgccaca



acatcgaggacggcagcgtgcagctcgccgaccactaccagc



agaacacccccatcggcgacggccccgtgctgctgcccgaca



accactacctgagcacccagtccgccctgagcaaagacccca



acgagaagcgcgatcacatggtcctgctggagttcgtgaccg



ccgccgggatcactctcggcatggacgagctgtacaagtaat



agggtaccggtcgacctgcagaagcttgcctcgagcagcgct



gctcgagagatctggatcataatcagccataccacatttgta



gaggttttacttgctttaaaaaacctcccacacctccccctg



aacctgaaacataaaatgaatgcaattgttgttgttaacttg



tttattgcagcttataatggttacaaataaagcaatagcatc



acaaatttcacaaataaagcatttttttcactgcattctagt



tgtggtttgtccaaactcatcaatgtatcttatcatgtctgg



taaccattctccaggttgagccagaccaatttgatggtagat



ttagcaaataaaaatacaggacacccagttaaatgtgaattt



ccgatgaacagcaaatacttttttagtattaaaaaagttcac



atttaggctcacgcctgtaatcccagcactttgggaggccga



ggcaggcagatcacctgaggtcaggagttcgagaccagcctg



gccaacatggtgaaaccccatctccactaaaaataccaaaaa



ttagccaggcgtgctggtgggcacctgtagttccagctactc



aggaggctaaggcaggagaattgcttgaacctgggaggcaga



ggttgcagtgagctgagatcgcaccattgcactctagcctgg



gcgacaagaacaaaactccatctcaaaaaaaaaaaaaaaaaa



aaagttcacatttaactgggcattctgtatttaattggtaat



ctgagatggcagggaacagcatcagcatggtgtgagggatag



gcattttttcattgtgtacagcttgtaaatcagtatttttaa



aactcaaagttaatggcttgggcatatttagaaaagagttgc



cgcacggacttgaaccctgtattcctaaaatctaggatcttg



ttctgatggtctgcacaactggctgggggtgtccagccactg



tccctcttgcctgggctccccagggcagttctgtcagcctct



ccatttccattcctgttccagcaaaacccaactgatagcaca



gcagcatttcagcctgtctacctctgtgcccacatacctgga



tgtctaccagccagaaaggtggcttagatttggttcctgtgg



gtggattatggcccccagaacttccctgtgcttgctgggggt



gtggagtggaaagagcaggaaatgggggaccctccgatactc



tatgggggtcctccaagtctctttgtgcaagttagggtaata



atcaatatggagctaagaaagagaaggggaactatgctttag



aacaggacactgtgccaggagcattgcagaaattatatggtt



ttcacgacagttctttttggtaggtactgttattatcctcag



tttgcagatgaggaaactgagacccagaaaggttaaataact



tgctagggtcacacaagtcataactgacaaagcctgattcaa



acccaggtctccctaacctttaaggtttctatgacgccagct



ctcctagggagtttgtcttcagatgtcttggctctaggtgtc



aaaaaaagacttggtgtcaggcaggcataggttcaagtccca



actctgtcacttaccaactgtgactaggtgattgaactgacc



atggaacctggtcacatgcaggagcaggatggtgaagggttc



ttgaaggcacttaggcaggacatttaggcaggagagaaaacc



tggaaacagaagagctgtctccaaaaatacccactggggaag



caggttgtcatgtgggccatgaatgggacctgttctggggta



accacgtgcggaccgagcggccgcaggaacccctagtgatgg



agttggccactccctctctgcgcgctcgctcgctcactgagg



ccgggcgaccaaaggtcgcccgacgcccgggctttgcccggg



cggcctcagtgagcgagcgagcgcgcagctgcctgcaggggc



gcctgatgcggtattttctccttacgcatctgtgcggtattt



cacaccgcatacgtcaaagcaaccatagtacgcgccctgtag



cggcgcattaagcgcggcgggtgtggtggttacgcgcagcgt



gaccgctacacttgccagcgccttagcgcccgctcctttcgc



tttcttcccttcctttctcgccacgttcgccggctttccccg



tcaagctctaaatcgggggctccctttagggttccgatttag



tgctttacggcacctcgaccccaaaaaacttgatttgggtga



tggttcacgtagtgggccatcgccctgatagacggtttttcg



ccctttgacgttggagtccacgttctttaatagtggactctt



gttccaaactggaacaacactcaactctatctcgggctattc



ttttgatttataagggattttgccgatttcggtctattggtt



aaaaaatgagctgatttaacaaaaatttaacgcgaattttaa



caaaatattaacgtttacaattttatggtgcactctcagtac



aatctgctctgatgccgcatagttaagccagccccgacaccc



gccaacacccgctgacgcgccctgacgggcttgtctgctccc



ggcatccgcttacagacaagctgtgaccgtctccgggagctg



catgtgtcagaggttttcaccgtcatcaccgaaacgcgcgag



acgaaagggcctcgtgatacgcctatttttataggttaatgt



catgataataatggtttcttagacgtcaggtggcacttttcg



gggaaatgtgcgcggaacccctatttgtttatttttctaaat



acattcaaatatgtatccgctcatgagacaataaccctgata



aatgcttcaataatattgaaaaaggaagagtatgagtattca



acatttccgtgtcgcccttattcccttttttgcggcattttg



ccttcctgtttttgctcacccagaaacgctggtgaaagtaaa



agatgctgaagatcagttgggtgcacgagtgggttacatcga



actggatctcaacagcggtaagatccttgagagttttcgccc



cgaagaacgttttccaatgatgagcacttttaaagttctgct



atgtggcgcggtattatcccgtattgacgccgggcaagagca



actcggtcgccgcatacactattctcagaatgacttggttga



gtactcaccagtcacagaaaagcatcttacggatggcatgac



agtaagagaattatgcagtgctgccataaccatgagtgataa



cactgcggccaacttacttctgacaacgatcggaggaccgaa



ggagctaaccgcttttttgcacaacatgggggatcatgtaac



tcgccttgatcgttgggaaccggagctgaatgaagccatacc



aaacgacgagcgtgacaccacgatgcctgtagcaatggcaac



aacgttgcgcaaactattaactggcgaactacttactctagc



ttcccggcaacaattaatagactggatggaggcggataaagt



tgcaggaccacttctgcgctcggcccttccggctggctggtt



tattgctgataaatctggagccggtgagcgtgggtctcgcgg



tatcattgcagcactggggccagatggtaagccctcccgtat



cgtagttatctacacgacggggagtcaggcaactatggatga



acgaaatagacagatcgctgagataggtgcctcactgattaa



gcattggtaactgtcagaccaagtttactcatatatacttta



gattgatttaaaacttcatttttaatttaaaaggatctaggt



gaagatcctttttgataatctcatgaccaaaatcccttaacg



tgagttttcgttccactgagcgtcagaccccgtagaaaagat



caaaggatcttcttgaaatcctttttttctgcgcgtaatctg



ctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttg



tttgccggatcaagagctaccaactctttttccgaaggtaac



tggcttcagcagagcgcagataccaaatactgttcttctagt



gtagccgtagttaggccaccacttcaagaactctgtagcacc



gcctacatacctcgctctgctaatcctgttaccagtggctgc



tgccagtggcgataagtcgtgtcttaccgggttggactcaag



acgatagttaccggataaggcgcagcggtcgggctgaacggg



gggttcgtgcacacagcccagcttggagcgaacgacctacac



cgaactgagatacctacagcgtgagctatgagaaagcgccac



gcttcccgaagggagaaaggcggacaggtatccggtaagcgg



cagggtcggaacaggagagcgcacgagggagcttccaggggg



aaacgcctggtatctttatagtcctgtcgggtttcgccacct



ctgacttgagcgtcgatttttgtgatgctcgtcaggggggcg



gagcctatggaaaaacgccagcaacgcggcctttttacggtt



cctggccttttgctggccttttgctcacatgtcctgcaggcag





GENE CASSETTE
59


AG004 OCCURS AT
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


BP 1 THROUGH
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


4438 OF SEQ ID
agagggagtggccaactccatcactaggggttcctgcggccg


NO: 34
cacgcgttacgtaatatttattgaagtttaatattgtgtttg



tgatacagaagtatttgctttaattctaaataaaaattttat



gcttttattgctggtttaagaagatttggattatccttgtac



tttgaggagaagtttcttatttgaaatattttggaaacaggt



cttttaatgtggaaagatagatattaatctcctcttctatta



ctctccaagatccaacaaaagtgattataccccccaaaatat



gatggtagtatcttatactaccatcattttataggcataggg



ctcttagctgcaaataatggaactaactctaataaagcagaa



cgcaaatattgtaaatattagagagctaacaatctctgggat



ggctaaaggatggagcttggaggctacccagccagtaacaat



attccgggctccactgttgaatggagacactacaactgcctt



ggatgggcagagatattatggatgctaagccccaggtgctac



cattaggacttctaccactgtccctaacgggtggagcccatc



acatgcctatgccctcactgtaaggaaatgaagctactgttg



tatatcttgggaagcacttggattaattgttatacagttttg



ttgaagaagacccctagggtaagtagccataactgcacacta



aatttaaaattgttaatgagtttctcaaaaaaaatgttaagg



ttgttagctggtatagtatatatcttgcctgttttccaagga



cttctttgggcagtaccttgtctgtgctggcaagcaactgag



acttaatgaaagagtattggagatatgaatgaattgatgctg



tatactctcagagtgccaaacatataccaatggacaagaagg



tgaggcagagagcagacaggcattagtgacaagcaaagatat



gcagaatttcattctcagcaaatcaaaagtcctcaacctggt



tggaagaatattggcactgaatggtatcaataaggttgctag



agagggttagaggtgcacaatgtgcttccataacattttata



cttctccaatcttagcactaatcaaacatggttgaatacttt



gtttactataactcttacagagttataagatctgtgaagaca



gggacagggacaatacccatctctgtctggttcataggtggt



atgtaatagatatttttaaaaataagtgagttaatgaatgag



ggtgagaatgaaggcacagaggtattagggggaggtgggccc



cagagaatggtgccaaggtccagtggggtgactgggatcagc



tcaggcctgacgctggccactcccacctagctcctttctttc



taatctgttctcattctccttgggaaggattgaggtctctgg



aaaacagccaaacaactgttatgggaacagcaagcccaaata



aagccaagcatcagggggatctgagagctgaaagcaacttct



gttccccctccctcagctgaaggggtggggaagggctcccaa



agccataactccttttaagggatttagaaggcataaaaaggc



ccctggctgagaacttccttcttcattctgcagttggtgaat



tccccggggatcctctagagtcgaaattcgccaccatggtga



gcaagggcgaggagctgttcaccggggtggtgcccatcctgg



tcgagctggacggcgacgtaaacggccacaagttcagcgtgt



ccggcgagggcgagggcgatgccacctacggcaagctgaccc



tgaagttcatctgcaccaccggcaagctgcccgtgccctggc



ccaccctcgtgaccaccctgacctacggcgtgcagtgcttca



gccgctaccccgaccacatgaagcagcacgacttcttcaagt



ccgccatgcccgaaggctacgtccaggagcgcaccatcttct



tcaaggacgacggcaactacaagacccgcgccgaggtgaagt



tcgagggcgacaccctggtgaaccgcatcgagctgaagggca



tcgacttcaaggaggacggcaacatcctggggcacaagctgg



agtacaactacaacagccacaacgtctatatcatggccgaca



agcagaagaacggcatcaaggtgaacttcaagatccgccaca



acatcgaggacggcagcgtgcagctcgccgaccactaccagc



agaacacccccatcggcgacggccccgtgctgctgcccgaca



accactacctgagcacccagtccgccctgagcaaagacccca



acgagaagcgcgatcacatggtcctgctggagttcgtgaccg



ccgccgggatcactctcggcatggacgagctgtacaagtaat



agggtaccggtcgacctgcagaagcttgcctcgagcagcgct



gctcgagagatctggatcataatcagccataccacatttgta



gaggttttacttgctttaaaaaacctcccacacctccccctg



aacctgaaacataaaatgaatgcaattgttgttgttaacttg



tttattgcagcttataatggttacaaataaagcaatagcatc



acaaatttcacaaataaagcatttttttcactgcattctagt



tgtggtttgtccaaactcatcaatgtatcttatcatgtctgg



taaccattctccaggttgagccagaccaatttgatggtagat



ttagcaaataaaaatacaggacacccagttaaatgtgaattt



ccgatgaacagcaaatacttttttagtattaaaaaagttcac



atttaggctcacgcctgtaatcccagcactttgggaggccga



ggcaggcagatcacctgaggtcaggagttcgagaccagcctg



gccaacatggtgaaaccccatctccactaaaaataccaaaaa



ttagccaggcgtgctggtgggcacctgtagttccagctactc



aggaggctaaggcaggagaattgcttgaacctgggaggcaga



ggttgcagtgagctgagatcgcaccattgcactctagcctgg



gcgacaagaacaaaactccatctcaaaaaaaaaaaaaaaaaa



aaagttcacatttaactgggcattctgtatttaattggtaat



ctgagatggcagggaacagcatcagcatggtgtgagggatag



gcattttttcattgtgtacagcttgtaaatcagtatttttaa



aactcaaagttaatggcttgggcatatttagaaaagagttgc



cgcacggacttgaaccctgtattcctaaaatctaggatcttg



ttctgatggtctgcacaactggctgggggtgtccagccactg



tccctcttgcctgggctccccagggcagttctgtcagcctct



ccatttccattcctgttccagcaaaacccaactgatagcaca



gcagcatttcagcctgtctacctctgtgcccacatacctgga



tgtctaccagccagaaaggtggcttagatttggttcctgtgg



gtggattatggcccccagaacttccctgtgcttgctgggggt



gtggagtggaaagagcaggaaatgggggaccctccgatactc



tatgggggtcctccaagtctctttgtgcaagttagggtaata



atcaatatggagctaagaaagagaaggggaactatgctttag



aacaggacactgtgccaggagcattgcagaaattatatggtt



ttcacgacagttctttttggtaggtactgttattatcctcag



tttgcagatgaggaaactgagacccagaaaggttaaataact



tgctagggtcacacaagtcataactgacaaagcctgattcaa



acccaggtctccctaacctttaaggtttctatgacgccagct



ctcctagggagtttgtcttcagatgtcttggctctaggtgtc



aaaaaaagacttggtgtcaggcaggcataggttcaagtccca



actctgtcacttaccaactgtgactaggtgattgaactgacc



atggaacctggtcacatgcaggagcaggatggtgaagggttc



ttgaaggcacttaggcaggacatttaggcaggagagaaaacc



tggaaacagaagagctgtctccaaaaatacccactggggaag



caggttgtcatgtgggccatgaatgggacctgttctggggta



accacgtgcggaccgagcggccgcaggaacccctagtgatgg



agttggccactccctctctgcgcgctcgctcgctcactgagg



ccgggcgaccaaaggtcgcccgacgcccgggctttgcccggg



cggcctcagtgagcgagcgagcgcgcag










Plasmid AG006 Composition








5′ ITR
 2



occurs @ bp 1 through bp 119 of SEQ ID



NO: 35





Human VMD2
12


Promoter
occurs @ bp 134 through bp 761 of SEQ ID



NO: 35





Added Kozak
 5



occurs @ bp 795 through 800 of SEQ ID NO:



34





E-GFP
24



occurs @ bp 801 through bp 1520 of SEQ ID



NO: 35





SV40 POLYA
 8



occurs at bp 1578 through bp 1813 of SEQ



ID NO: 35





RLBP1 INTRONIC
14


SEQUENCE AS
occurs at bp 1821 through bp 3323 of SEQ


STUFFER
ID NO: 35


SEQUENCE





3′ ITR
 9



occurs at bp 3352 through bp 3481 of SEQ



ID NO: 35





AMP BACTERIAL
15


BACKBONE
occurs at bp 3482 through bp 6100 of SEQ



ID NO: 35





Sequence of
35


plasmid AG006
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc



gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag



agagggagtggccaactccatcactaggggttcctgcggccg



cacgcgttacgtaattctgtcattttactagggtgatgaaat



tcccaagcaacaccatccttttcagataagggcactgaggct



gagagaggagctgaaacctacccggcgtcaccacacacaggt



ggcaaggctgggaccagaaaccaggactgttgactgcagccc



ggtattcattctttccatagcccacagggctgtcaaagaccc



cagggcctagtcagaggctcctccttcctggagagttcctgg



cacagaagttgaagctcagcacagccccctaacccccaactc



tctctgcaaggcctcaggggtcagaacactggtggagcagat



cctttagcctctggattttagggccatggtagagggggtgtt



gccctaaattccagccctggtctcagcccaacaccctccaag



aagaaattagaggggccatggccaggctgtgctagccgttgc



ttctgagcagattacaagaagggactaagacaaggactcctt



tgtggaggtcctggcttagggagtcaagtgacggcggctcag



cactcacgtgggcagtgccagcctctaagagtgggcaggggc



actggccacagagtcccagggagtcccaccagcctagtcgcc



agaccgaattccccggggatcctctagagtcgaaattcgcca



ccatggtgagcaagggcgaggagctgttcaccggggtggtgc



ccatcctggtcgagctggacggcgacgtaaacggccacaagt



tcagcgtgtccggcgagggcgagggcgatgccacctacggca



agctgaccctgaagttcatctgcaccaccggcaagctgcccg



tgccctggcccaccctcgtgaccaccctgacctacggcgtgc



agtgcttcagccgctaccccgaccacatgaagcagcacgact



tcttcaagtccgccatgcccgaaggctacgtccaggagcgca



ccatcttcttcaaggacgacggcaactacaagacccgcgccg



aggtgaagttcgagggcgacaccctggtgaaccgcatcgagc



tgaagggcatcgacttcaaggaggacggcaacatcctggggc



acaagctggagtacaactacaacagccacaacgtctatatca



tggccgacaagcagaagaacggcatcaaggtgaacttcaaga



tccgccacaacatcgaggacggcagcgtgcagctcgccgacc



actaccagcagaacacccccatcggcgacggccccgtgctgc



tgcccgacaaccactacctgagcacccagtccgccctgagca



aagaccccaacgagaagcgcgatcacatggtcctgctggagt



tcgtgaccgccgccgggatcactctcggcatggacgagctgt



acaagtaatagggtaccggtcgacctgcagaagcttgcctcg



agcagcgctgctcgagagatctggatcataatcagccatacc



acatttgtagaggttttacttgctttaaaaaacctcccacac



ctccccctgaacctgaaacataaaatgaatgcaattgttgtt



gttaacttgtttattgcagcttataatggttacaaataaagc



aatagcatcacaaatttcacaaataaagcatttttttcactg



cattctagttgtggtttgtccaaactcatcaatgtatcttat



catgtctggtaaccattctccaggttgagccagaccaatttg



atggtagatttagcaaataaaaatacaggacacccagttaaa



tgtgaatttccgatgaacagcaaatacttttttagtattaaa



aaagttcacatttaggctcacgcctgtaatcccagcactttg



ggaggccgaggcaggcagatcacctgaggtcaggagttcgag



accagcctggccaacatggtgaaaccccatctccactaaaaa



taccaaaaattagccaggcgtgctggtgggcacctgtagttc



cagctactcaggaggctaaggcaggagaattgcttgaacctg



ggaggcagaggttgcagtgagctgagatcgcaccattgcact



ctagcctgggcgacaagaacaaaactccatctcaaaaaaaaa



aaaaaaaaaaaagttcacatttaactgggcattctgtattta



attggtaatctgagatggcagggaacagcatcagcatggtgt



gagggataggcattttttcattgtgtacagcttgtaaatcag



tatttttaaaactcaaagttaatggcttgggcatatttagaa



aagagttgccgcacggacttgaaccctgtattcctaaaatct



aggatcttgttctgatggtctgcacaactggctgggggtgtc



cagccactgtccctcttgcctgggctccccagggcagttctg



tcagcctctccatttccattcctgttccagcaaaacccaact



gatagcacagcagcatttcagcctgtctacctctgtgcccac



atacctggatgtctaccagccagaaaggtggcttagatttgg



ttcctgtgggtggattatggcccccagaacttccctgtgctt



gctgggggtgtggagtggaaagagcaggaaatgggggaccct



ccgatactctatgggggtcctccaagtctctttgtgcaagtt



agggtaataatcaatatggagctaagaaagagaaggggaact



atgctttagaacaggacactgtgccaggagcattgcagaaat



tatatggttttcacgacagttctttttggtaggtactgttat



tatcctcagtttgcagatgaggaaactgagacccagaaaggt



taaataacttgctagggtcacacaagtcataactgacaaagc



ctgattcaaacccaggtctccctaacctttaaggtttctatg



acgccagctctcctagggagtttgtcttcagatgtcttggct



ctaggtgtcaaaaaaagacttggtgtcaggcaggcataggtt



caagtcccaactctgtcacttaccaactgtgactaggtgatt



gaactgaccatggaacctggtcacatgcaggagcaggatggt



gaagggttcttgaaggcacttaggcaggacatttaggcagga



gagaaaacctggaaacagaagagctgtctccaaaaataccca



ctggggaagcaggttgtcatgtgggccatgaatgggacctgt



tctggggtaaccacgtgcggaccgagcggccgcaggaacccc



tagtgatggagttggccactccctctctgcgcgctcgctcgc



tcactgaggccgggcgaccaaaggtcgcccgacgcccgggct



ttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcc



tgcaggggcgcctgatgcggtattttctccttacgcatctgt



gcggtatttcacaccgcatacgtcaaagcaaccatagtacgc



gccctgtagcggcgcattaagcgcggcgggtgtggtggttac



gcgcagcgtgaccgctacacttgccagcgccttagcgcccgc



tcctttcgctttcttcccttcctttctcgccacgttcgccgg



ctttccccgtcaagctctaaatcgggggctccctttagggtt



ccgatttagtgctttacggcacctcgaccccaaaaaacttga



tttgggtgatggttcacgtagtgggccatcgccctgatagac



ggtttttcgccctttgacgttggagtccacgttctttaatag



tggactcttgttccaaactggaacaacactcaactctatctc



gggctattcttttgatttataagggattttgccgatttcggt



ctattggttaaaaaatgagctgatttaacaaaaatttaacgc



gaattttaacaaaatattaacgtttacaattttatggtgcac



tctcagtacaatctgctctgatgccgcatagttaagccagcc



ccgacacccgccaacacccgctgacgcgccctgacgggcttg



tctgctcccggcatccgcttacagacaagctgtgaccgtctc



cgggagctgcatgtgtcagaggttttcaccgtcatcaccgaa



acgcgcgagacgaaagggcctcgtgatacgcctatttttata



ggttaatgtcatgataataatggtttcttagacgtcaggtgg



cacttttcggggaaatgtgcgcggaacccctatttgtttatt



tttctaaatacattcaaatatgtatccgctcatgagacaata



accctgataaatgcttcaataatattgaaaaaggaagagtat



gagtattcaacatttccgtgtcgcccttattcccttttttgc



ggcattttgccttcctgtttttgctcacccagaaacgctggt



gaaagtaaaagatgctgaagatcagttgggtgcacgagtggg



ttacatcgaactggatctcaacagcggtaagatccttgagag



ttttcgccccgaagaacgttttccaatgatgagcacttttaa



agttctgctatgtggcgcggtattatcccgtattgacgccgg



gcaagagcaactcggtcgccgcatacactattctcagaatga



cttggttgagtactcaccagtcacagaaaagcatcttacgga



tggcatgacagtaagagaattatgcagtgctgccataaccat



gagtgataacactgcggccaacttacttctgacaacgatcgg



aggaccgaaggagctaaccgcttttttgcacaacatggggga



tcatgtaactcgccttgatcgttgggaaccggagctgaatga



agccataccaaacgacgagcgtgacaccacgatgcctgtagc



aatggcaacaacgttgcgcaaactattaactggcgaactact



tactctagcttcccggcaacaattaatagactggatggaggc



ggataaagttgcaggaccacttctgcgctcggcccttccggc



tggctggtttattgctgataaatctggagccggtgagcgtgg



gtctcgcggtatcattgcagcactggggccagatggtaagcc



ctcccgtatcgtagttatctacacgacggggagtcaggcaac



tatggatgaacgaaatagacagatcgctgagataggtgcctc



actgattaagcattggtaactgtcagaccaagtttactcata



tatactttagattgatttaaaacttcatttttaatttaaaag



gatctaggtgaagatcctttttgataatctcatgaccaaaat



cccttaacgtgagttttcgttccactgagcgtcagaccccgt



agaaaagatcaaaggatcttcttgaaatcctttttttctgcg



cgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc



ggtggtttgtttgccggatcaagagctaccaactctttttcc



gaaggtaactggcttcagcagagcgcagataccaaatactgt



tcttctagtgtagccgtagttaggccaccacttcaagaactc



tgtagcaccgcctacatacctcgctctgctaatcctgttacc



agtggctgctgccagtggcgataagtcgtgtcttaccgggtt



ggactcaagacgatagttaccggataaggcgcagcggtcggg



ctgaacggggggttcgtgcacacagcccagcttggagcgaac



gacctacaccgaactgagatacctacagcgtgagctatgaga



aagcgccacgcttcccgaagggagaaaggcggacaggtatcc



ggtaagcggcagggtcggaacaggagagcgcacgagggagct



tccagggggaaacgcctggtatctttatagtcctgtcgggtt



tcgccacctctgacttgagcgtcgatttttgtgatgctcgtc



aggggggcggagcctatggaaaaacgccagcaacgcggcctt



tttacggttcctggccttttgctggccttttgctcacatgtc



ctgcaggcag





GENE CASSETTE
60


OF PLASMID
ctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggc


AG006 OCCURS AT
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcag


BP 1 THROUGH
agagggagtggccaactccatcactaggggttcctgcggccg


3481 OF SEQ ID
cacgcgttacgtaattctgtcattttactagggtgatgaaat


NO: 35
tcccaagcaacaccatccttttcagataagggcactgaggct



gagagaggagctgaaacctacccggcgtcaccacacacaggt



ggcaaggctgggaccagaaaccaggactgttgactgcagccc



ggtattcattctttccatagcccacagggctgtcaaagaccc



cagggcctagtcagaggctcctccttcctggagagttcctgg



cacagaagttgaagctcagcacagccccctaacccccaactc



tctctgcaaggcctcaggggtcagaacactggtggagcagat



cctttagcctctggattttagggccatggtagagggggtgtt



gccctaaattccagccctggtctcagcccaacaccctccaag



aagaaattagaggggccatggccaggctgtgctagccgttgc



ttctgagcagattacaagaagggactaagacaaggactcctt



tgtggaggtcctggcttagggagtcaagtgacggcggctcag



cactcacgtgggcagtgccagcctctaagagtgggcaggggc



actggccacagagtcccagggagtcccaccagcctagtcgcc



agaccgaattccccggggatcctctagagtcgaaattcgcca



ccatggtgagcaagggcgaggagctgttcaccggggtggtgc



ccatcctggtcgagctggacggcgacgtaaacggccacaagt



tcagcgtgtccggcgagggcgagggcgatgccacctacggca



agctgaccctgaagttcatctgcaccaccggcaagctgcccg



tgccctggcccaccctcgtgaccaccctgacctacggcgtgc



agtgcttcagccgctaccccgaccacatgaagcagcacgact



tcttcaagtccgccatgcccgaaggctacgtccaggagcgca



ccatcttcttcaaggacgacggcaactacaagacccgcgccg



aggtgaagttcgagggcgacaccctggtgaaccgcatcgagc



tgaagggcatcgacttcaaggaggacggcaacatcctggggc



acaagctggagtacaactacaacagccacaacgtctatatca



tggccgacaagcagaagaacggcatcaaggtgaacttcaaga



tccgccacaacatcgaggacggcagcgtgcagctcgccgacc



actaccagcagaacacccccatcggcgacggccccgtgctgc



tgcccgacaaccactacctgagcacccagtccgccctgagca



aagaccccaacgagaagcgcgatcacatggtcctgctggagt



tcgtgaccgccgccgggatcactctcggcatggacgagctgt



acaagtaatagggtaccggtcgacctgcagaagcttgcctcg



agcagcgctgctcgagagatctggatcataatcagccatacc



acatttgtagaggttttacttgctttaaaaaacctcccacac



ctccccctgaacctgaaacataaaatgaatgcaattgttgtt



gttaacttgtttattgcagcttataatggttacaaataaagc



aatagcatcacaaatttcacaaataaagcatttttttcactg



cattctagttgtggtttgtccaaactcatcaatgtatcttat



catgtctggtaaccattctccaggttgagccagaccaatttg



atggtagatttagcaaataaaaatacaggacacccagttaaa



tgtgaatttccgatgaacagcaaatacttttttagtattaaa



aaagttcacatttaggctcacgcctgtaatcccagcactttg



ggaggccgaggcaggcagatcacctgaggtcaggagttcgag



accagcctggccaacatggtgaaaccccatctccactaaaaa



taccaaaaattagccaggcgtgctggtgggcacctgtagttc



cagctactcaggaggctaaggcaggagaattgcttgaacctg



ggaggcagaggttgcagtgagctgagatcgcaccattgcact



ctagcctgggcgacaagaacaaaactccatctcaaaaaaaaa



aaaaaaaaaaaagttcacatttaactgggcattctgtattta



attggtaatctgagatggcagggaacagcatcagcatggtgt



gagggataggcattttttcattgtgtacagcttgtaaatcag



tatttttaaaactcaaagttaatggcttgggcatatttagaa



aagagttgccgcacggacttgaaccctgtattcctaaaatct



aggatcttgttctgatggtctgcacaactggctgggggtgtc



cagccactgtccctcttgcctgggctccccagggcagttctg



tcagcctctccatttccattcctgttccagcaaaacccaact



gatagcacagcagcatttcagcctgtctacctctgtgcccac



atacctggatgtctaccagccagaaaggtggcttagatttgg



ttcctgtgggtggattatggcccccagaacttccctgtgctt



gctgggggtgtggagtggaaagagcaggaaatgggggaccct



ccgatactctatgggggtcctccaagtctctttgtgcaagtt



agggtaataatcaatatggagctaagaaagagaaggggaact



atgctttagaacaggacactgtgccaggagcattgcagaaat



tatatggttttcacgacagttctttttggtaggtactgttat



tatcctcagtttgcagatgaggaaactgagacccagaaaggt



taaataacttgctagggtcacacaagtcataactgacaaagc



ctgattcaaacccaggtctccctaacctttaaggtttctatg



acgccagctctcctagggagtttgtcttcagatgtcttggct



ctaggtgtcaaaaaaagacttggtgtcaggcaggcataggtt



caagtcccaactctgtcacttaccaactgtgactaggtgatt



gaactgaccatggaacctggtcacatgcaggagcaggatggt



gaagggttcttgaaggcacttaggcaggacatttaggcagga



gagaaaacctggaaacagaagagctgtctccaaaaataccca



ctggggaagcaggttgtcatgtgggccatgaatgggacctgt



tctggggtaaccacgtgcggaccgagcggccgcaggaacccc



tagtgatggagttggccactccctctctgcgcgctcgctcgc



tcactgaggccgggcgaccaaaggtcgcccgacgcccgggct



ttgcccgggcggcctcagtgagcgagcgagcgcgcag










Plasmid TM042 Composition








ΔITR
 1



occurs at bp 4 through bp 106 of SEQ ID



NO: 50





Human RLBP1
 3


Promoter(short)
Occurs at bp 119 through bp 708 of SEQ ID



NO: 50





MODIFIED
 4


SV40INTRON
occurs at bp 723 through bp 905 of SEQ ID



NO: 50





Added Kozak
 5



occurs at bp 919 through bp 924 of SEQ ID



NO: 50





HUMAN RLBP1
 6


GENE CDS
occurs at bp 925 through bp 1878 of SEQ ID



NO: 50





SV40 POLYA
 8



occurs at bp 1937 through bp 2172 of SEQ



ID NO: 50





3′ ITR
 9



occurs at bp 2201 through bp 2330 of SEQ



ID NO: 50





KAN-R BACTERIAL
49


BACKBONE
occurs at bp 2331 through bp 4989 of SEQ



ID NO: 50





Sequence of
50


plasmid TM042
ctgcgcgctcgctcgctcactgaggccgcccgggcaaagccc



gggcgtcgggcgacctttggtcgcccggcctcagtgagcgag



cgagcgcgcagagagggagtggggtaccacgcgtttgtcctc



tccctgcttggccttaaccagccacatttctcaactgacccc



actcactgcagaggtgaaaactaccatgccaggtcctgctgg



ctgggggaggggtgggcaataggcctggatttgccagagctg



ccactgtagatgtagtcatatttacgatttcccttcacctct



tattaccctggtggtggtggtgggggggggggggtgctctct



cagcaaccccaccccgggatcttgaggagaaagagggcagag



aaaagagggaatgggactggcccagatcccagccccacagcc



gggcttccacatggccgagcaggaactccagagcaggagcac



acaaaggagggctttgatgcgcctccagccaggcccaggcct



ctcccctctcccctttctctctgggtcttcctttgccccact



gagggcctcctgtgagcccgatttaacggaaactgtgggcgg



tgagaagttccttatgacacactaatcccaacctgctgaccg



gaccacgcctccagcggagggaacctctagagctccaggaca



ttcaggtaccaggtagccccaaggaggagctgccgaatcgat



ggatcgggaactgaaaaaccagaaagttaactggtaagttta



gtctttttgtcttttatttcaggtcccggatccggtggtggt



gcaaatcaaagaactgctcctcagtggatgttgcctttactt



ctaggcctgtacggaagtgttacttctgctctaaaagctgcg



gaattgtacccgccccgggatccatcgattgaattcgccacc



atgtcagaaggggtgggcacgttccgcatggtacctgaagag



gaacaggagctccgtgcccaactggagcagctcacaaccaag



gaccatggacctgtctttggcccgtgcagccagctgccccgc



cacaccttgcagaaggccaaggatgagctgaacgagagagag



gagacccgggaggaggcagtgcgagagctgcaggagatggtg



caggcgcaggcggcctcgggggaggagctggcggtggccgtg



gcggagagggtgcaagagaaggacagcggcttcttcctgcgc



ttcatccgcgcacggaagttcaacgtgggccgtgcctatgag



ctgctcagaggctatgtgaatttccggctgcagtaccctgag



ctctttgacagcctgtccccagaggctgtccgctgcaccatt



gaagctggctaccctggtgtcctctctagtcgggacaagtat



ggccgagtggtcatgctcttcaacattgagaactggcaaagt



caagaaatcacctttgatgagatcttgcaggcatattgcttc



atcctggagaagctgctggagaatgaggaaactcaaatcaat



ggcttctgcatcattgagaacttcaagggctttaccatgcag



caggctgctagtctccggacttcagatctcaggaagatggtg



gacatgctccaggattccttcccagcccggttcaaagccatc



cacttcatccaccagccatggtacttcaccacgacctacaat



gtggtcaagcccttcttgaagagcaagctgcttgagagggtc



tttgtccacggggatgacctttctggtttctaccaggagatc



gatgagaacatcctgccctctgacttcgggggcacgctgccc



aagtatgatggcaaggccgttgctgagcagctctttggcccc



caggcccaagctgagaacacagccttctgaggatcgtaccgg



tcgacctgcagaagcttgcctcgagcagcgctgctcgagaga



tctggatcataatcagccataccacatttgtagaggttttac



ttgctttaaaaaacctcccacacctccccctgaacctgaaac



ataaaatgaatgcaattgttgttgttaacttgtttattgcag



cttataatggttacaaataaagcaatagcatcacaaatttca



caaataaagcatttttttcactgcattctagttgtggtttgt



ccaaactcatcaatgtatcttatcatgtctggtaaccacgtg



cggaccgagcggccgcaggaacccctagtgatggagttggcc



actccctctctgcgcgctcgctcgctcactgaggccgggcga



ccaaaggtcgcccgacgcccgggctttgcccgggcggcctca



gtgagcgagcgagcgcgcagctgcctgcagggttccatccca



atggcgcgtcaattcactggccgtcgttttacaacgtcgtga



ctgggaaaaccctggcgttacccaacttaatcgccttgcagc



acatccccctttcgccagctggcgtaatagcgaagaggcccg



caccgatcgcccttcccaacagttgcgcagcctgaatggcga



atggcgcctgatgcggtattttctccttacgcatctgtgcgg



tatttcacaccgcatatggtgcactctcagtacaatctgctc



tgatgccgcatagttaagccagccccgacacccgccaacacc



cgctgacgcgccctgacgggcttgtctgctcccggcatccgc



ttacagacaagctgtgaccgtctccgggagctgcatgtgtca



gaggttttcaccgtcatcaccgaaacgcgcgagacgaaaggg



cctcgtgatacgcctatttttataggttaatgtcatgataat



aatggtttcttagacgtcaggtggcacttttcggggaaatgt



gcgcggaacccctatttgtttatttttctaaatacattcaaa



tatgtatccgctcatgagacaataaccctgataaatgcttca



ataatattgaaaaaggaagagtatgagccatattcaacggga



aacgtcttgctctaggccgcgattaaattccaacatggatgc



tgatttatatgggtataaatgggctcgcgataatgtcgggca



atcaggtgcgacaatctatcgattgtatgggaagcccgatgc



gccagagttgtttctgaaacatggcaaaggtagcgttgccaa



tgatgttacagatgagatggtcagactaaactggctgacgga



atttatgcctcttccgaccatcaagcattttatccgtactcc



tgatgatgcatggttactcaccactgcgatccctgggaaaac



agcattccaggtattagaagaatatcctgattcaggtgaaaa



tattgttgatgcgctggcagtgttcctgcgccggttgcattc



gattcctgtttgtaattgtccttttaacagcgatcgcgtatt



tcgtctcgctcaggcgcaatcacgaatgaataacggtttggt



tgatgcgagtgattttgatgacgagcgtaatggctggcctgt



tgaacaagtctggaaagaaatgcataaacttttgccattctc



accggattcagtcgtcactcatggtgatttctcacttgataa



ccttatttttgacgaggggaaattaataggttgtattgatgt



tggacgagtcggaatcgcagaccgataccaggatcttgccat



cctatggaactgcctcggtgagttttctccttcattacagaa



acggctttttcaaaaatatggtattgataatcctgatatgaa



taaattgcagtttcatttgatgctcgatgagtttttctaact



gtcagaccaagtttactcatatatactttagattgatttaaa



acttcatttttaatttaaaaggatctaggtgaagatcctttt



tgataatctcatgaccaaaatcccttaacgtgagttttcgtt



ccactgagcgtcagaccccgtagaaaagatcaaaggatcttc



ttgagatcctttttttctgcgcgtaatctgctgcttgcaaac



aaaaaaaccaccgctaccagcggtggtttgtttgccggatca



agagctaccaactctttttccgaaggtaactggcttcagcag



agcgcagataccaaatactgttcttctagtgtagccgtagtt



aggccaccacttcaagaactctgtagcaccgcctacatacct



cgctctgctaatcctgttaccagtggctgctgccagtggcga



taagtcgtgtcttaccgggttggactcaagacgatagttacc



ggataaggcgcagcggtcgggctgaacggggggttcgtgcac



acagcccagcttggagcgaacgacctacaccgaactgagata



cctacagcgtgagctatgagaaagcgccacgcttcccgaagg



gagaaaggcggacaggtatccggtaagcggcagggtcggaac



aggagagcgcacgagggagcttccagggggaaacgcctggta



tctttatagtcctgtcgggtttcgccacctctgacttgagcg



tcgatttttgtgatgctcgtcaggggggcggagcctatggaa



aaacgccagcaacgcggcctttttacggttcctggccttttg



ctggccttttgctcacatgttctttcctgcgttatcccctga



ttctgtggataaccgtattaccgcctttgagtgagctgatac



cgctcgccgcagccgaacgaccgagcgcagcgagtcagtgag



cgaggaagcggaagagcgcccaatacgcaaaccgcctctccc



cgcgcgttggccgattcattaatgcagctggcacgacaggtt



tcccgactggaaagcgggcagtgagcgcaacgcaattaatgt



gagttagctcactcattaggcaccccaggctttacactttat



gcttccggctcgtatgttgtgtggaattgtgagcggataaca



atttcacacaggaaacagctatgaccatgattacgccaagct



cggcgcgccattgggatggaaccctgcaggcag





GENE CASSETTE
61


TM042 OCCURS AT
cgcgctcgctcgctcactgaggccgcccgggcaaagcccggg


BP 4 THROUGH
cgtcgggcgacctttggtcgcccggcctcagtgagcgagcga


2330 OF SEQ ID
gcgcgcagagagggagtggggtaccacgcgtttgtcctctcc


NO: 50
ctgcttggccttaaccagccacatttctcaactgaccccact



cactgcagaggtgaaaactaccatgccaggtcctgctggctg



ggggaggggtgggcaataggcctggatttgccagagctgcca



ctgtagatgtagtcatatttacgatttcccttcacctcttat



taccctggtggtggtggtgggggggggggggtgctctctcag



caaccccaccccgggatcttgaggagaaagagggcagagaaa



agagggaatgggactggcccagatcccagccccacagccggg



cttccacatggccgagcaggaactccagagcaggagcacaca



aaggagggctttgatgcgcctccagccaggcccaggcctctc



ccctctcccctttctctctgggtcttcctttgccccactgag



ggcctcctgtgagcccgatttaacggaaactgtgggcggtga



gaagttccttatgacacactaatcccaacctgctgaccggac



cacgcctccagcggagggaacctctagagctccaggacattc



aggtaccaggtagccccaaggaggagctgccgaatcgatgga



tcgggaactgaaaaaccagaaagttaactggtaagtttagtc



tttttgtcttttatttcaggtcccggatccggtggtggtgca



aatcaaagaactgctcctcagtggatgttgcctttacttcta



ggcctgtacggaagtgttacttctgctctaaaagctgcggaa



ttgtacccgccccgggatccatcgattgaattcgccaccatg



tcagaaggggtgggcacgttccgcatggtacctgaagaggaa



caggagctccgtgcccaactggagcagctcacaaccaaggac



catggacctgtctttggcccgtgcagccagctgccccgccac



accttgcagaaggccaaggatgagctgaacgagagagaggag



acccgggaggaggcagtgcgagagctgcaggagatggtgcag



gcgcaggcggcctcgggggaggagctggcggtggccgtggcg



gagagggtgcaagagaaggacagcggcttcttcctgcgcttc



atccgcgcacggaagttcaacgtgggccgtgcctatgagctg



ctcagaggctatgtgaatttccggctgcagtaccctgagctc



tttgacagcctgtccccagaggctgtccgctgcaccattgaa



gctggctaccctggtgtcctctctagtcgggacaagtatggc



cgagtggtcatgctcttcaacattgagaactggcaaagtcaa



gaaatcacctttgatgagatcttgcaggcatattgcttcatc



ctggagaagctgctggagaatgaggaaactcaaatcaatggc



ttctgcatcattgagaacttcaagggctttaccatgcagcag



gctgctagtctccggacttcagatctcaggaagatggtggac



atgctccaggattccttcccagcccggttcaaagccatccac



ttcatccaccagccatggtacttcaccacgacctacaatgtg



gtcaagcccttcttgaagagcaagctgcttgagagggtcttt



gtccacggggatgacctttctggtttctaccaggagatcgat



gagaacatcctgccctctgacttcgggggcacgctgcccaag



tatgatggcaaggccgttgctgagcagctctttggcccccag



gcccaagctgagaacacagccttctgaggatcgtaccggtcg



acctgcagaagcttgcctcgagcagcgctgctcgagagatct



ggatcataatcagccataccacatttgtagaggttttacttg



ctttaaaaaacctcccacacctccccctgaacctgaaacata



aaatgaatgcaattgttgttgttaacttgtttattgcagctt



ataatggttacaaataaagcaatagcatcacaaatttcacaa



ataaagcatttttttcactgcattctagttgtggtttgtcca



aactcatcaatgtatcttatcatgtctggtaaccacgtgcgg



accgagcggccgcaggaacccctagtgatggagttggccact



ccctctctgcgcgctcgctcgctcactgaggccgggcgacca



aaggtcgcccgacgcccgggctttgcccgggcggcctcagtg



agcgagcgagcgcgcag
















TABLE 3







Plasmid Construction








SEQUENCE



IDENTIFIER


(SEQ. ID. NO:)
Construction summary










Plasmid TM017








1
PvuII/MluI restriction fragment of Δ5′ ITR


ΔITR
element cloned into PvuII/MluI restriction



fragment of plasmid backbone


3
Blunted BamHI/MluI restriction fragment of


Human RLBP1
human RLBP1 promoter (short) was cloned


Promoter(short)
into blunted SacII/MluI restriction



fragment of plasmid backbone


4
MluI/ClaI restriction fragment a clone


MODIFIED
containing an hCMV promoter and modified


SV40INTRON
SV40 intron was cloned into MluI/ClaI



restriction fragment of plasmid backbone.



The hCMV promoter was removed during



subsequent cloning to insert human RLBP1



promoter (short)


5, 6
EcoRI/AgeI restriction fragment containing


Added Kozak
Kozak and human RLBP1 gene CDS was cloned


AND HUMAN
into EcoRI/AgeI restriction fragment of


RLBP1
plasmid backbone


GENE CDS


8
BglII/BstEII restriction fragment of SV40


SV40 POLYA
polyA was cloned into BglII/BsteII



restriction fragment of the plasmid



backbone


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS (Stratagene)







Plasmid TM037 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, AAV-MCS (Stratagene)


10
Blunted HindIII/EcoRI restriction fragment


Human RLBP1
of human RLBP1 promoter (long) was cloned


Promoter(long)
into blunted MluI/EcoRI restriction



fragment of plasmid backbone


5, 6
EcoRI/AgeI restriction fragment containing


Added Kozak
Kozak and human RLBP1 gene CDS cloned into


AND HUMAN
EcoRI/AgeI restriction fragment of plasmid


RLBP1
backbone


GENE CDS


8
BglII/BstEII restriction fragment


SV40 POLYA
containing SV40 polyA was cloned into



BglII/BsteII restriction fragment of the



plasmid backbone


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS (Stratagene)







Plasmid AG007 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, pAAV-MCS (Stratagene)


11
MluI/EcoRI restriction fragment containing


Human RPE65
human RPE65 promoter cloned into


Promoter
MluI/EcoRI restriction fragment of the



plasmid backbone


5, 6
EcoRI/AgeI restriction fragment containing


ADDED-KOZAK
Kozak and human RLBP1 gene CDS cloned into


and HUMAN RLBP1
EcoRI/AgeI restriction fragment of plasmid


GENE CDS
backbone


8
BglII/BstEII restriction fragment


SV40 POLYA
containing SV40 polyA was cloned into



BglII/BsteII restriction fragment of the



plasmid backbone


14
BstEII restriction fragment containing


RLBP1 INTRONIC
RLBP1 intron1 stuffer sequence was cloned


SEQUENCE AS
into BstEII restriction fragment of the


STUFFER
plasmid backbone


SEQUENCE


9
Present in original Amp resistant


3′ ITR
backbone, pAAV-MCS (Stratagene)







Plasmid TM039 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, AAV-MCS purchased from



Stratagene


22
EcoRI/MluI restriction fragment containing


CMV-enhancer
CMV-enhancer with CBA promoter was cloned


with CBA
into EcoRI/MluI restriction fragment of


promoter
plasmid backbone


5, 6
EcoRI/SalI restriction fragment containing


Added Kozak
Kozak and human RLBP1 gene CDS was cloned


AND HUMAN
into EcoRI/SalI restriction fragment of


RLBP1
plasmid backbone


GENE CDS


8
BglII/BstEII restriction fragment


SV40 POLYA
containing SV40 polyA was cloned into



BglII/BsteII restriction fragment of the



plasmid backbone


23 REVERSE
Plasmid backbone was cut with BstEII then


COMPLEMENT OF
blunted. The stuffer was PCR amplified


RLBP1 INTRON
from human cell line (HEK293 or ARPE19)


STUFFER
genomic DNA, the product was



phosphorylated and ligated into backbone.


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS (Stratagene)







Plasmid TM040 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, AAV-MCS (Stratagene)


3
Blunted BamHI/MluI restriction fragment of


Human RLBP1
human RLBP1 promoter (short) was cloned


promoter
into blunted SacII/MluI restriction


(short)
fragment of plasmid backbone


4
MluI/ClaI restriction fragment containing


MODIFIED
an hCMV promoter and modified SV40 intron


SV40INTRON
was cloned into MluI/ClaI restriction



fragment of plasmid backbone. The hCMV



promoter was removed during subsequent



cloning to insert human RLBP1 promoter



(short)


5, 6
EcoRI/SalI restriction fragment containing


Added Kozak
Kozak and human RLBP1 gene CDS cloned into


AND HUMAN
EcoRI/SalI restriction fragment of plasmid


RLBP1
backbone


GENE CDS


8
BglII/BstEII restriction fragment


SV40 POLYA
containing SV40 polyA was cloned into



BglII/BsteII restriction fragment of the



plasmid backbone


23 REVERSE
Plasmid backbone was cut with BstEII then


COMPLEMENT OF
blunted. The stuffer was PCR amplified


RLBP1 INTRON
from human cell line (HEK293 or ARPE19)


STUFFER
genomic DNA, the product was



phosphorylated and ligated into backbone.


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS purchased from



Stratagene







Plasmid TM016 construction summary








1
PvuII/MluI restriction of Δ5′ ITR element


Δ5′ ITR
cloned into PvuII/MluI restriction



fragment of plasmid backbone


3
Blunted BamHI/MluI restriction fragment of


Human RLBP1
human RLBP1 promoter (short) was cloned


promoter
into blunted SacII/MluI restriction


(short)
fragment of plasmid backbone


4
MluI/ClaI restriction fragment of


MODIFIED
containing an hCMV promoter and modified


SV40INTRON
SV40 intron was cloned into MluI/ClaI



restriction fragment of plasmid backbone.



The hCMV promoter was removed during



subsequent cloning to insert human RLBP1



promoter (short)


24
EcoRI/Age fragment containing GFP was


E_GFP
blunted then cloned into the SalI digested



and blunted backbone


8
BglII/BstEII restriction fragment


SV40 POLYA
containing SV40 polyA was cloned into



BglII/BsteII restriction fragment of the



plasmid backbone


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS purchased from



Stratagene







Plasmid TM035 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, AAV-MCS purchased from



Stratagene


10
Blunted HindIII/EcoRI restriction fragment


Human RLBP1
of human RLBP1 promoter (long) was cloned


promoter (long)
into blunted MluI/EcoRI restriction



fragment of plasmid backbone


24
EcoRI/Age I digested fragment containing


E_GFP
GFP was blunted then cloned into the SalI



digested and blunted backbone


8
BglII/BstEII restriction fragment


SV40 POLYA
containing SV40 polyA was cloned into



BglII/BstEII restriction fragment of the



plasmid backbone


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS purchased from



Stratagene







Plasmid AG012 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, AAV-MCS purchased from



Stratagene


13
The plasmid backbone was digested with


SYNUCLEIN
MluI/AgeI.


INTRONIC
The synuclein stuffer was PCR amplified


SEQUENCE AS
from plasmid pBV5, the product was


STUFFER
digested with MluI/AgeI, phosphorylated


SEQUENCE
and ligated into the plasmid backbone.


8
BglII/BstEII restriction fragment from


SV40 POLYA
GeneArt synthesized clone containing SV40



polyA was cloned into BglII/BstEII



restriction fragment of the plasmid



backbone


14
BstEII restriction fragment from


RLBP1 INTRONIC
intermediary clone containing RLBP1


SEQUENCE AS
intron1 stuffer sequence cloned into


STUFFER
BstEII restriction fragment of the plasmid


SEQUENCE
backbone


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS purchased from



Stratagene







Plasmid AG004 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, AAV-MCS purchased from



Stratagene


11
MluI/EcoRI restriction fragment from


Human RPE65
GeneArt synthesized clone containing human


Promoter
RPE65 promoter cloned into MluI/EcoRI



restriction fragment of the plasmid



backbone


24
An EcoRI/AgeI digested fragment from an


E_GFP
intermediary clone was blunted then cloned



into the SalI digested and blunted



backbone


8
BglII/BstEII restriction fragment from


SV40 POLYA
GeneArt synthesized clone containing SV40



polyA was cloned into BglII/BstEII



restriction fragment of the plasmid



backbone


14
BstEII restriction fragment from


RLBP1 INTRONIC
intermediary clone containing RLBP1


SEQUENCE AS
intron1 stuffer sequence cloned into


STUFFER
BstEII restriction fragment of the plasmid


SEQUENCE
backbone


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS purchased from



Stratagene







Plasmid AG006 construction summary








2
Present in original Amp resistant


5′ ITR
backbone, AAV-MCS purchased from



Stratagene


12
MluI/EcoRI restriction fragment from


HUMAN VMD2
GeneArt synthesized clone containing human


PROMOTER
VMD2 promoter cloned into MluI/EcoRI



restriction fragment of the plasmid



backbone


24
An EcoRI/AgeI digested fragment from an


E_GFP
intermediary clone was blunted then cloned



into the SalI digested and blunted



backbone


8
BglII/BstEII restriction fragment from


SV40 POLYA
GeneArt synthesized clone containing SV40



polyA was cloned into BglII/BstEII



restriction fragment of the plasmid



backbone


14
BstEII restriction fragment from


RLBP1 INTRONIC
intermediary clone containing RLBP1


SEQUENCE AS
intron1 stuffer sequence cloned into


STUFFER
BstEII restriction fragment of the plasmid


SEQUENCE
backbone


9
Present in original Amp resistant


3′ ITR
backbone, AAV-MCS purchased from



Stratagene







Plasmid TM042 Construction Summary








1-ΔITR, 3-
SbfI restriction fragment of Plamsid


Human RLBP1
pTM017 was cloned into a SbfI restriction


Promoter(short),
fragment of Puc57 with kanamycin


4- MODIFIED
resistance gene backbone.


SV40INTRON,


5, 6- Added


Kozak AND


HUMAN RLBP1


GENE, 8- SV40


POLYA, and 9-


3′ ITR
















TABLE 4







Viral Vector Composition: Vector Genome and Caspid








SEQUENCE
SEQUENCE IDENTIFIER


ELEMENTS
(SEQ. ID. NO:)










Viral Vector NVS1 (Generated from plasmid TM017 or TM042,


and AAVRep2/Cap2 plasmid)


The viral vector contains a self complementary genome with


the following genomic elements in the 5′ to 3′ direction


packaged in the viral capsid AAV2.








SC5′ ITR
36


Reverse
62


Complementary


sequence of


SV40polyA


Reverse
63


Complementary


sequence of


HUMAN RLBP1


GENE CDS


Reverse
64


Complementary


sequence of


Added KOZAK


Reverse
65


Complementary


sequence of


Modified


SV40INTRON


Reverse
66


Complementary


sequence of


Human RLBP1


PROMOTER


(short)


ΔITR
1


Human RLBP1
3


PROMOTER


(short)


Modified
4


SV40INTRON


Added Kozak
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS1








AAV2 CAPSID
19, 68 and 69


SEQUENCE
(Encoded by 18)







Viral Vector NVS2 (Generated from plasmid TM017 or TM042,


and AAVRep2/Cap8 plasmid)


The viral vector contains a self complementary genome with


the following genomic elements in the 5′ to 3′ direction


packaged in the viral capsid AAV8.








SC5′ ITR
36


Reverse
62


Complementary


sequence of


SV40polyA


Reverse
63


Complementary


sequence of


HUMAN RLBP1


GENE CDS


Reverse
64


Complementary


sequence of


Added KOZAK


Reverse
65


Complementary


sequence of


Modified


SV40INTRON


Reverse
66


Complementary


sequence of


Human RLBP1


PROMOTER


(short)


ΔITR
1


Human RLBP1
3


PROMOTER


(short)


Modified
4


SV40INTRON


Added Kozak
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS2








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector NVS3 (Generated from plasmid TM037 and


AAVRep2/Cap2 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RLBP1
10


PROMOTER (long)


ADDED KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS3








AAV2 CAPSID
19, 68 and 69


SEQUENCE
(Encoded by 18)







Viral Vector NVS4 (Generated from plasmid TM037 and


AAVRep2/Cap8 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RLBP1
10


PROMOTER (long)


ADDED KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS4








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector NVS5 (Generated from plasmid AG007 and


AAVRep2/Cap2 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RPE65
11


PROMOTER


ADDED-KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


RLBP1 INTRONIC
14


SEQUENCE AS


STUFFER


SEQUENCE


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS5








AAV2 CAPSID
19, 68 and 69


SEQUENCE
(Encoded by 18)







Viral Vector NVS6 (Generated from plasmid AG007 and


AAVRep2/Cap8 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RPE65
11


PROMOTER


ADDED-KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


RLBP1 INTRONIC
14


SEQUENCE AS


STUFFER


SEQUENCE


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS6








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector NVS7 (Generated from plasmid TM039 and


AAVRep2/Cap2 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


CMV Enhancer
22


and CBA


PROMOTER


(GENEBANK


ACCESSION


DD215332 FROM


BP 1 to BP 161)


ADDED KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


REVERSE
23


COMPLEMENT OF


RLBP1 INTRONIC


SEQUENCE AS


STUFFER


SEQUENCE (NT


010274.17)


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS7








AAV2 CAPSID
19, 68 and 69


SEQUENCE
(Encoded by 18)







Viral Vector NVS8 (Generated from plasmid TM039 and


AAVRep2/Cap8 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


CMV Enhancer
22


and CBA


PROMOTER


(GENEBANK


ACCESSION


DD215332 FROM


BP 1 to BP 161)


ADDED KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


REVERSE
23


COMPLEMENT OF


RLBP1 INTRONIC


SEQUENCE AS


STUFFER


SEQUENCE (NT


010274.17)


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS8








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector NVS9 (Generated from plasmid TM040 and


AAVRep2/Cap2 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RLBP1
3


PROMOTER


(short)


MODIFIED SV40
4


INTRON


ADDED KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


REVERSE
23


COMPLEMENT OF


RLBP1 INTRONIC


SEQUENCE AS


STUFFER


SEQUENCE


(NT_010274.17)


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS9








AAV2 CAPSID
19, 68 and 69


SEQUENCE
(Encoded by 18)







Viral Vector NVS10 (Generated from plasmid TM040 and


AAVRep2/Cap8 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RLBP1
3


PROMOTER


(short)


MODIFIED SV40
4


INTRON


ADDED KOZAK
5


HUMAN RLBP1
6


GENE CDS


SV40 POLYA
8


REVERSE
23


COMPLEMENT OF


RLBP1 INTRONIC


SEQUENCE AS


STUFFER


SEQUENCE


(NT_010274.17)


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS10








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral vector scAAV8-pRLBP1(short)-eGFP


(eGFP Reporter viral vector generated from plasmid TM016


and AAVRep2/Cap8 plasmid)


The viral vector contains a self complementary genome with


the following genomic elements in the 5′ to 3′ direction


packaged in the viral capsid AAV8.








SC5′ ITR
36


Reverse
62


Complementary


sequence of


SV40polyA


Reverse
67


Complementary


sequence of


eGFP


Reverse
64


Complementary


sequence of


Added KOZAK


Reverse
65


Complementary


sequence of


Modified


SV40INTRON


Reverse
66


Complementary


sequence of


Human RLBP1


PROMOTER


(short)


ΔITR
1


HUMAN RLBP1
3


PROMOTER


(short)


MODIFIED SV40
4


INTRON


ADDED KOZAK
5


eGFP
24


SV40 POLYA
8


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF Viral vector scAAV8-


pRLBP1(short)-eGFP








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector AAV8-pRLBP1(long)-eGFP


(eGFP Reporter viral vector generated from plasmid TM035


and AAVRep2/Cap8 plasmid) The viral vector genome contains


the following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RLBP1
10


PROMOTER (long)


ADDED KOZAK
5


eGFP
24


SV40 POLYA
8


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF Viral Vector


AAV8-pRLBP1(long)-eGFP








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector AAV8-pRPE65-eGFP


(eGFP Reporter viral vector generated from plasmid AG004


and AAVRep2/Cap8 plasmid) The viral vector genome contains


the following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN RPE65
11


PROMOTER


ADDED KOZAK
5


eGFP
24


SV40 POLYA
8


RLBP1 INTRONIC
14


SEQUENCE AS


STUFFER


SEQUENCE


(NT_010274.17)


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF Viral Vector AAV8-pRPE65-eGFP








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector AAV8-pVMD2-eGFP


(eGFP Reporter viral vector generated from plasmid AG006


and AAVRep2/Cap8 plasmid) The viral vector genome contains


the following genomic elements in the 5′ to 3′ direction








5′ ITR
2


HUMAN VMD2
12


PROMOTER


ADDED KOZAK
5


eGFP
24


SV40 POLYA
8


RLBP1 INTRONIC
14


SEQUENCE AS


STUFFER


SEQUENCE


(NT_010274.17)


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF Viral Vector AAV8-pVMD2-eGFP








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)







Viral Vector NVS11 (Generated from plasmid AG012 and


AAVRep2/Cap8 plasmid) The viral vector genome contains the


following genomic elements in the 5′ to 3′ direction








5′ ITR
2


SYNUCLEIN
13


INTRONIC


SEQUENCE AS


STUFFER


SEQUENCE


SV40 POLYA
8


RLBP1 INTRONIC
14


SEQUENCE AS


STUFFER


SEQUENCE


(NT_010274.17)


3′ ITR
9







CAPSID PROTEIN SEQUENCE OF NVS11








AAV8 CAPSID
21, 70, and 71


SEQUENCE
(Encoded by 20)









Example 2
Subretinal Injection of rAAV Vectors in Mice

2.1 Subretinal Injection of rAAV Vectors in Mice


Subretinal injection of an rAAV vector can achieve efficient transduction of RPE and other retinal cells because subretinal injection induces a bleb of concentrated virus in intimate contact with RPE cells and the neural retina. In addition, the subretinal space has a relatively high degree of immunoprivilege and typically very little evidence of inflammation is seen in the vicinity of the injection site. Thus, subretinal injection was a preferred route for delivery of rAAV vectors in mouse retina. However, other routes of delivery may be used, for example, intravitreal injection.


Supplies/reagents:

Leica M844 F40 Ophthalmic Surgical Microscope


1% cyclopentolate: Bausch & Lomb Cat#965911


2.5%-10% phenylephrine: Altaire Pharmaceuticals Cat#05626


0.5% Proparacaine: Bausch & Lomb Cat#NDC 54799-500-12


10 μl Hamilton syringe: VWR Cat#89184-476


33G blunt-ended needle: Hamilton Cat#7803-05


Fluorescein sodium salt: Sigma Cat#F6377


Test Articles Used in this Example:


scAAV8-pRLBP1(short)-eGFP viral vector 1×109 vg/eye


AAV8-pRLBP1(long)-eGFP viral vector 1×109 vg/eye


AAV8-pRPE-eGFP viral vector 1×109 vg/eye


AAV8-VMD2-eGFP viral vector 1×109 vg/eye


Protocol:

The subretinal injection was performed either in both eyes or unilaterally in the right eye. All procedures were performed under aseptic conditions, using sterile reagents, syringes and appropriate personal protection equipment.


Subretinal Injection Procedures:





    • The mouse pupils were dilated by 1 drop of 1% cyclopentolate and followed by 1 drop of 2.5%-10% phenylephrine

    • The mouse was anesthetized by using Avertin (250 mg/kg) i.p. and a drop of 0.5% Proparacine topically (local anesthetic) in the eye

    • An approximately 0.5 mm incision was made nasally, posterior to the limbus with a microscalpel

    • The blunt-ended needle on the 10 μl Hamilton syringe was inserted tangentially through the scleral incision toward the temporal retina. The needle was advanced until resistance was felt. The 1 μl of diluted rAAV vector (containing fluorescein with the concentration of 1:50) was then injected slowly into the subretinal space, and the needle is withdrawn through the incision

    • The eye was examined and the success of the subretinal injection was confirmed by visualization of a bleb containing fluorescein. The success of injection and the degree of retinal damage (hemorrhage) were scored.

    • An antibiotic ointment was applied to the eye immediately after the injection


      2.2. rAAV Vectors Induced GFP Expression and its Cell-Type Specifics in Mouse Retina





To study the rAAV vector-induced gene transduction and cell-type specifics in the mouse retina, the eGFP expression in retinal cross sections and RPE/retina flatmounts were examined. One approach used to identify the eGFP expressing cell types was to co-label eGFP positive cells with retinal cell markers by immunocytochemistry staining in cryosections.


Supplies/Reagents:
Primary Antibodies for Immunocytochemistry Staining:





    • Anti-CRALBP antibody: Thermo cat#MA1-813

    • Anti-GFAP antibody: Covance cat#SMI-21

    • Anti-Opsin Blue antibody: Millipore cat#AB 5407

    • Anti-Opsin Red antibody: Millipore cat#AB5405

    • Anti-Vimentin antibody: Santa Cruz cat#sc-7557

    • Anti-PKC α antibody: C-20 Santa Cruz cat#sc -208





Secondary Antibodies for Immunocytochemistry Staining:





    • Goat anti-mouse IgG: Invitrogen Cat#A11005

    • Goat anti-rat IgG: Invitrogen Cat#A11007

    • Donkey anti-rabbit IgG: Invitrogen Cat#A21207





Other Supplies/Reagents:





    • Vectashield Mounting Medium with DAPI: Vector Laboratories, Burlingame Cat#H-1200),

    • Zeiss Imaging system, AxioVision Software

    • Zeiss LSM 510 confocal microscope, ZEN version of the Zeiss software Protocol:





The mouse eyeball was removed and placed in 4% PFA (paraformaldehyde) for 2 hours at 25° C. and then in PBS buffer for 1-3 days in 4° C. till dissection. The cornea, lens and vitreous were removed from the eye ball and the retinal and RPE/choroid was flatmounted with Vectashield mounting medium on to the slide. The GFP expression in flatmount was captured by Zeiss Imaging system and quantified using AxioVision Software. After imaging, the slides with retinal flatmounts were placed in 0.25% triton buffer at 25° C. for 30 min and then the retinal flatmounts were removed from the slides. The eGFP positive areas of the retina flatmounts were cut and embedded in OCT and then cryosectioned. The immunocytochemistry staining using retinal cell markers was applied in the cryosections. The images were captured by Zeiss LSM 510 confocal microscope and ZEN version of the Zeiss software.


The Immunocytochemistry Staining Procedures:
Day 1.





    • air dry sections at room temperature 1 hour.

    • place slides in PBS+0.25% Triton 15 min×2

    • block in 1% BSA+PBS+0.25% Triton 90 min

    • incubate slides with primary antibody in 1% BSA+PBS+0.25% Triton at 4° C. overnight





Day 2.





    • take out slides from 4° C., leave them at 25° C. for 30 min

    • wash slides in PBS+0.25% Triton 15 min×2

    • incubate slides with secondary 1:800 at 25° C. for 90 min

    • wash slides in PBS+0.25% Triton 15 min×2

    • mount slides with Vectashield Mounting Medium with DAPI












TABLE 5







The retinal cell markers and dilutions used in the study











Cell Type
Cell Marker
Dilutions







Müller cell
Anti-CRALBP
 1:1000




Anti-Vimentin
1:100




Anti-GFAP
 1:1000



Photoreceptor
Anti-Opsin Red/Green
1:250




Anti-Opsin Blue
1:250



Neuron in INL
Anti-PKCα
1:200



Astrocytes
Anti-GFAP
 1:1000

















TABLE 6







Immunohistochemistry results that describe the transduction of cell types by test viral vectors.














scAAV8-
AAV8-
AAV8-
AAV8


Cell Type
Cell Marker
pRLBP1(short)-eGFP
pRLBP1(long)-eGFP
pRPE65-eGFP
pVMD2-eGFP





RPE

+
+
+
+


Müller cell
CRALBP
+
+





Vimentin
+
+





GFAP
+
+




Photoreceptor
Opsin Red/Green

+
+
+



Opsin Blue


+
+



Recoverin
ND
ND
+
ND


Neuron in INL
PKCα






Ganglion Cell
NeuN

ND
ND
ND


Astrocytes
GFAP









+, indicates expression of GFP in a given cell type


−, no GFP expression


ND, Not Determined






Results:





    • All tested viral vectors were functional in the mouse retina.

    • scAAV8-pRLBP1(short)-eGFP vector leads to selective expression of GFP in RPE and the Müller cells in the neural retina.

    • AAV8-pRLBP1(long)-eGFP leads to expression of GFP in RPE, Müller cells and photoreceptors in the neural retina.

    • AAV8-pRPE65-eGFP and AAV8-pVMD2-eGFP lead to GFP expression in RPE and photoreceptors in the neural retina.





CONCLUSION

These results demonstrate that the combination of promoter, AAV genome conformation and AAV capsid sequence can lead to different transduction properties in specific cell types, to achieve the desired effect. Expression of the RLBP1 gene product in RPE and Müller cells of the retina, represents the desired on-target cell type expression. RLBP1 short promoter packaged in a self-complementary genome in conjunction with an AAV8 serotype capsid induces gene expression in RPE and Müller cells in the neural retina without off-target cell expression.


The RLBP1-long promoter packaged in a single-stranded genome in conjunction with an AAV8 serotype capsid induces gene expression in RPE and Müller cells, which are on-target cell types, and also in photoreceptors, which is an off-target cell type.


The RPE65 and VMD2 promoter packaged in a single-stranded genome in conjunction with an AAV8 serotype capsid induces gene expression in RPE cells but also in photoreceptors, which is an off-target cell type.


Example 3
mRNA Based Assay to Measure Vector-Mediated Expression of a Human RLBP1 Transgene Relative to Endogenous Mouse RLBP1 mRNA Expression

The expression levels and tissue specificity of an rAAV-transduced transgene will vary depending on the vector serotype, the vector genome, the tissue-specific promoter used and the dose injected. A goal of gene replacement therapy is to achieve a level of expression that is sufficient to compensate for the missing endogenous gene expression while not over expressing the gene to toxic levels.


An assay has been developed to measure the vector-mediated expression of human RLBP1 mRNA relative to the endogenous levels of mouse RLBP1 mRNA following subretinal injections of various AAV vectors at different doses in wild-type mice. This assay utilized Taqman® Gene Expression Assays containing primers and probes for specifically detecting human or mouse RLBP1 cDNA. Prior to performing the experiment the Taqman® Gene Expression Assays were tested for species specificity using plasmid DNA containing either human or mouse RLBP1 cDNA sequences. In brief, Taqman® reagents were used to co-amplify either mouse or human RLBP1 cDNA with mouse GAPDH cDNA as an endogenous control. The levels of the mouse or human RLBP1 were normalized to the internal GAPDH control and then these normalized levels were compared with one another.


Supplies/reagents:





    • RNA extraction
      • Qiagen RNeasy micro kit (Qiagen cat #74004)
      • Qiagen RNase-Free DNase Set (Qiagen cat#79254)
      • Beta-Mercaptoethanol (Sigma cat#63689)
      • Qiagen Stainless-Steel 5 mm beads (Qiagen cat#69989)
      • 2.0 ml Seal Rite Microcentrifuge tube (USA Scientific cat#1620-2700)
      • Qiagen TissueLyser II (cat#85300)

    • cDNA Synthesis
      • High Capacity cDNA Reverse Transcription Kit (Applied Biosystems cat#4368814)
      • RNase Inhibitor (Applied Biosystems cat#N8080119)
      • BioRad Thermal cycler

    • Relative Quantitation PCR
      • 2× TaqMan® Universal PCR Master Mix (Applied Biosystems cat#4304437)
      • 20× TaqMan® Gene Expression Assay for human RLBP1 (Applied Biosystems cat#4331182: Hs00165632.m1)
      • 20× TaqMan® Gene Expression Assay for mouse RLBP1 (Applied Biosystems cat#4331182: Mm00445129.m1)
      • 20× Applied Biosystems® Mouse GAPD (GAPDH) Endogenous Control (VIC®/MGB Probe, Primer Limited) (Applied Biosystems cat#4352339E)
      • Applied Biosystems Real-Time PCR machine model 7900HT.

    • Test articles used in this example:
      • NVS8 viral vector
      • NVS10 viral vector
      • NVS4 viral vector
      • NVS2 viral vector
      • NVS6 viral vector





Protocol:

At the termination of the in vivo experiment neural retina was dissected out of the eyes, placed in a 2 ml microcentrifuge tube and flash frozen on dry ice. The remaining eye cup (minus retina and lens) was frozen in a separate tube. Samples were stored at −80° C. until RNA isolation. Total RNA was extracted using a Qiagen RNeasy micro kit with DNase treatment. For tissue homogenization and lysis, a Qiagen TissueLyzer was used. In particular, a 5 mm stainless-steel bead was added to each tissue-containing tube while on dry ice. Samples were transferred to room temperature and 350 μl of buffer RLT containing 1% beta-mercaptoethanol was added. Samples were processed on the TissueLyzer with a shaking frequency of 30 Hz for two 2 minute cycles. The standard Qiagen RNeasy micro kit protocol for RNA extraction with DNase treatment was then followed with one minor modification. Prior to elution the RNA column was allowed to air dry for >10 minutes to ensure elimination of residual ethanol. Total RNA was stored at −80° C. until ready for cDNA synthesis.


Total RNA concentration was determined using a Nanodrop spectrophotometer. Each sample was adjusted to a final concentration of 50 ng/μl. cDNA was generated using the Applied Biosystems High Capacity cDNA reverse transcriptase kit. A master mix of reagents from the High Capacity cDNA RT kit was prepared such that each 10 μl contained 2 μl of 10× High Capacity RT buffer, 0.8 μl of 25× dNTPs (100 mM), 2 μl of Reverse Transcriptase random primers, 0.4 μl of RNase inhibitor, 1 μl of Multiscribe Reverse transcriptase and 3.8 μl of RNAse-free water. 10 μl of the 50 ng/μl stock of each total RNA was dispensed into a well of a 96-well PCR amplification plate and then 10 μl of the RT master mix was added to each well. The plate was placed in a Bio-Rad thermal cycler and operated using the following parameters: 25° C. for 10 min, 37° C. for 120 min., 85° C. for 5 min then hold at 4 degrees until terminate program. cDNA was stored at −20° C. prior to Relative quantitative PCR reaction set-up.


The cDNA concentration was adjusted to a final concentration of 20 ng/μl by adding 5 μl of RNAse-free water to each well of the cDNA reaction (this is based on the initial total RNA concentration and assuming 100% conversion to cDNA). For each cDNA sample set up two different multiplex qPCR reactions; one using the mouse RLBP1 Taqman Expression Assay probes with the mouse GAPDH endogenous control, and the other using the human RLBP1 Taqman Expression Assay probes with the mouse GAPDH endogenous control. Each of these two reactions were performed in duplicate for each sample. For each sample, 5 μl of the 20 ng/μl cDNA sample was dispensed into a well of a 385-well plate. Two separate master mixes were prepared, one for the mouse RLBP1 Taqman assay and one for the human RLBP1 assay such that each 15 μl of mixture contained 10 μl of 2× TaqMan®Universal PCR Master Mix, 1 μl of 20× TaqMan® Gene Expression Assay for either mouse or human RLBP1, 1 μl of 20× Applied Biosystems® Mouse GAPD (GAPDH) Endogenous Control, and 3 μl of RNAse-free water. 15 μl of the appropriate master mix was dispensed into the well containing the cDNA. The plate was placed in an ABI 7900HT Real Time PCR machine and run using the relative quantitation program with the following parameters: an initial incubation at 50° C. for 2 min then 40 cycles of the following two steps, 15 sec. at 95° C. and 1 min. at 60° C.


The relative quantitation plate results were imported into a RQ study document using the ABI RQ Manager 1.2. The data were analyzed using the automatic threshold setting to generate average and average ΔCt which is the difference in Ct readings of the RLBP1 cDNA (mouse or human) minus the Ct of the internal endogenous GAPDH. The data were exported into Microsoft Excel and used to calculate the ΔΔCt value by subtracting the mouse RLBP1 ΔCt value from the human RLBP1 ΔCt for each sample. The relative expression was calculated using the calculation 2−ΔΔCt this expresses the relative expression of human RLBP1 as a fold change of the mouse endogenous RLBP1 expression. To portray the results as expression of human RLBP1 as a percent of the mouse endogenous expression the relative expression value was multiplied by 100.


Results: mRNA Expression.



FIG. 1A illustrates that NVS8, NVS4, NVS2 and NVS6 successfully transduce both the neural retina cells and the RPE cells in the posterior eyecup. Vector NVS10 transduces the RPE cells but barely at the level of detection limit in the neural retina.



FIG. 1B illustrates that NVS2 is the only vector to show mRNA expression in the neural retina at a lower dose of 1×108 vg/eye.


CONCLUSION

These surprising results demonstrated that the specific combination of promoter, AAV genome conformation, and AAV capsid sequence can lead to different transduction properties in different cell types in the retina. In general, all tested vectors successfully lead to vector-mediated human RLBP1 mRNA expression. More specifically, NVS2 is the most potent vector in expressing human RLBP1 mRNA in the RPE cells (in the posterior eyecup) and in the neural retina in both doses tested (1×109 and 1×108 vg/eye), while NVS4 and NVS6 lead to detectable vector-mediated human RLBP1 mRNA expression at the dose of 1×109 vg/eye, and only in the RPE at the dose of 1×108 vg/eye. NVS8 and NVS10 lead to detectable mRNA expression in the RPE and neural retina at the dose of 1×109 vg/eye but almost at the detection limit at the dose of 1×108 vg/eye.


Example 4
Electroretinogram-Based Dark Adaptation Assay

One approach for assessing treatments that modify the visual cycle is to quantify the recovery of visual function in the dark following a bright light exposure (i.e. dark adaptation). Dark adaptation after extensive light exposure is driven largely by the ability of the eye to regenerate photopigment via the visual cycle. Modifications to the visual cycle achieved through treatment will therefore lead to a change in the kinetics of dark adaptation.


An assay has been developed to monitor the recovery of visual function in mice that is based on quantifying dark adaptation using an electroretinogram (ERG). The ERG-based assay typically proceeds over two days with an initial baseline and subsequent follow-up measurement to assess recovery after exposure to light that bleaches a fraction of the photopigment (photobleach). This procedure developed for testing the invention first determines the maximum electrical response of each eye 5 ms after a flash of light during the a-wave portion of the ERG trace. The test subsequently compares the 5 ms a-wave amplitude 4 hours after a photobleach to assess the fraction of maximum amplitude recovered in that time. If the visual cycle is functioning normally, the ERG amplitude will approach baseline values in 4 hours. A delayed visual cycle will result in lower recovery of photopigment with a corresponding reduction in ERG a-wave amplitude recovery after photobleach.


Supplies/Reagents:





    • ERG system: Diagnosys, Espion E2 console with ColorDome full field ganzfeld stimulator

    • Ketamine

    • Xylazine

    • 2.5% phenylephrine

    • 1% cyclopentolate

    • 0.5% proparacaine

    • Active electrode: Gold loop contact lens electrode (Mayo, part number N30)

    • Reference electrode: Nasopharyngeal electrode (Grass, part number F-ERG-G)

    • Ground electrode: Platinum needle electrode (Grass, part number F-E2)

    • Hydrating drops: Novartis, Genteal Mild to Moderate

    • Syringe pump: Harvard Apparatus, part number Pump 11 Plus





Protocol:

Mice are placed in the dark overnight for approximately 20 hours before baseline ERGs are recorded. Immediately preceding recording, eyes are dilated with 1-2 drops of 1% cyclopentolate and 1-2 drops of 2.5% phenylephrine. 1-2 drops of 0.5% proparacaine (a topical anesthetic) are also applied. Mice are then anesthetized with an intraperitoneal injection of a cocktail of ketamine and xylazine (100-150 mg/kg and 5-10 mg/kg, respectively). Three electrodes are then placed to enable recording an ERG from one eye per mouse. The active electrode on the eye is a gold loop contact lens, the reference is a nasopharyngeal electrode placed in the mouth and the ground is a subdermal platinum needle electrode placed on the back just behind the head. Eyes are kept moist and electrical contact is maintained through continuous application of hydrating drops with a syringe pump (300 μl/hour). ERG amplitude is recorded by averaging the electrical response to three white flashes (2.7 log scotopic candela second per square meter) delivered by the xenon lamp in the ganzfeld dome. A-wave amplitude reported is the voltage measured 5 ms after the xenon flash as assessed using software analysis routines developed for this purpose (Mathworks, Matlab).


Dark adaptation is assessed by quantifying the ERG a-wave amplitude 4 hours after a photobleach. These experiments typically occur 48 hours after baseline determination. Mice are first housed in the dark overnight just as with the baseline measurements so that ERG recordings occur approximately 20 hours later. Eyes are dilated with 1-2 drops of 2.5% phenylephrine and 1-2 drops of 1% cyclopentolate immediately preceding photobleach. A sequence of 16 flashes of light (3.7 log scotopic candela second per square meter) is then delivered to the eye resulting in a photopigment bleach. Mice are placed back in the dark for 4 hours to recover visual function. ERGs are then recorded utilizing the same protocol used for the baseline determination. The recovery of visual function for each eye is defined as:






DA
=


a


-


wave





amplitude





4





hours





post


-


bleach


baseline





a


-


wave





amplitude







FIG. 2 illustrates the results of the assay when applied to RLBP1−/− and RLBP1+/+mice. RLBP1+/+mice exhibit nearly full recovery (up to 96%) 4 hours post-bleach. In contrast, RLBP1−/− mice recover minimal visual function (11%) at the same time point due to severely delayed visual cycle kinetics (Saari et al 2001). This 8-9 fold window between RLBP1+/+and −/− mice is the assay window achievable for testing vectors injected into RLBP1−/− mice.


Using the ERG-based dark adaptation assay described above, the improvement of dark adaptation efficiency is tested in RLBP1 knockout (KO) mice where therapeutic vectors are introduced subretinally. Since the subretinal injection involves the displacement of neural retina from the RPE, it is crucial to determine if the neural retina is reattached to the RPE to avoid false negative results for the test articles in the ERG assay. One week after subretinal injection of viral vectors into mouse eyes, optical coherence tomography (OCT) is performed to visualize the condition of the retina. Eyes with unresolved retinal detachment were excluded from ERG measurement.


At each time point, mice were dark adapted overnight (>12 hours) and the ERG a-wave amplitude from each eye was established as the maximum dark adapted response to light (100%). The fully dark adapted eyes were then exposed to a series of bright flashes (as described in previous section) and a-wave amplitude was quantified 4 hours later. The term “percentage of normal” is defined as the percentage of the second a-wave recovery measurement with respect to the value obtained from the maximum a-wave recovery measurement.


Positive efficacy, or efficacious effect, is defined as the difference between test measurement and negative (naïve) control being statistically significant at a given time point post-injection.


Test Articles Used in this Example Includes:

    • NVS1 viral vector
    • NVS2 viral vector
    • NVS3 viral vector
    • NVS4 viral vector
    • NVS5 viral vector
    • NVS11 viral vector



FIGS. 3A-D illustrate that viral vectors expressing RLBP1 improve the rate of dark adaptation in RLBP1 KO mice. Efficacy assessments were performed for each group vs. naïve controls with statistics calculated using a one way ANOVA with a Newman-Keuls multiple comparison test. The mean+3 standard deviations (SD) for naïve (uninjected) eyes and eyes receiving 1×109 vg/eye of the negative control AAV-null vector (NVS11) for all related studies are shown to indicate the approximate threshold for efficacy (a-wave recoveries above this line typically exhibit statistically significant efficacy). This approach for displaying the degree of efficacy is similar to that presented in gene therapy publications (Jacobson et al. 2006 and Roman et al. 2007).



FIG. 3A shows that at a dose of 3×108 vg/eye, NVS2 is efficacious in improving the rate of dark adaptation as early as 14 days post treatment, and the efficacy endures at least 350 days. A dose of 3×108 vg/eye of NVS4 is also efficacious for at least 30-204 days post-treatment. NVS2 at the dose of approximately 3×108 vg/eye has been tested in RLBP1 KO mouse model in 3 independent experiments. In each experiment at all time points tested up to 350 days post injection the vector demonstrated efficacy.



FIG. 3B shows that NVS1 at the same dose (3×108 vg/eye) demonstrated efficacy starting 84 days post-injection, with efficacy enduring to at least 350 days. NVS5 and NVS3 at the same dose did not demonstrate efficacy for up to 154 days post drug administration. Data presented in FIGS. 3A and 3B suggested that even though the viral vector genome is equivalent, the vector can be of different potency when packaged in different AAV capsid serotype (NVS1 versus NVS2). In addition, the specific combination of vector serotype, promoter, and vector genome conformation can affect the potency of the vector (NVS1 carries a self-complementary genome while NVS3 and NVS4 carry a single-stranded genome, all with different promoter sequences). This result further confirms that the combination of genome conformation and capsid serotype can affect the efficiency of recovery outcome.



FIG. 3C shows that, at the dose of 1×109 vg/eye, NVS2 is efficacious as early as 18 days post treatment, and the efficacy endures at least 375 days. At the dose of 1×109 vg/eye, NVS11, which is a negative control AAV-null vector, did not show significant difference in improvement of rate of dark adaptation when compared to uninjected control (individual data points not shown, but the historical mean+3SD line is displayed for comparison). A dose of 1×109 vg/eye of NVS4 is also efficacious for at least 30-204 days post-treatment.



FIG. 3D shows that at a dose of 3×109 vg/eye, NVS3 and NVS5, respectively, are efficacious in improving the rate of dark adaptation as early as day 26 post-treatment, and the efficacy endures at least 371 days.



FIG. 4A demonstrates that NVS2 at multiple doses is efficacious at improving the rate of dark adaptation for at least 94 days post-injection. Both the 3×108 and 1×109 vg/eye groups were efficacious compared to naïve controls based on a one way ANOVA with a Newman-Keuls multiple comparison test. FIG. 4B displays the data from FIG. 4A in a different format. In this case, the plot displays the percentage of eyes/group with an a-wave recovery greater than that defined by the mean+3SD of the naïve group from several experiments. The results indicate that for NVS2, 50% of 3×107 vg/eye treated eyes and 100% of 3×108 and 1×109 vg/eye treated eyes demonstrated efficacious a-wave recovery, and that a dose-response curve is established.



FIG. 5A demonstrates that NVS4 at multiple doses is efficacious at improving the rate of dark adaptation for at least 93 days post-injection. Both the 3×108 and 1×109 vg/eye groups were efficacious compared to naïve controls based on a one way ANOVA with a Newman-Keuls multiple comparison test. FIG. 5B displays the data from FIG. 5A in a different format. In this case, the plot displays the percentage of eyes/group with an a-wave recovery greater than that defined by the mean+3SD of the naïve group from several experiments. The results suggest that for NVS4, 85% of eyes treated with 3×108 and 1×109 vg/eye exhibited an increase in dark adaptation rate.



FIG. 6 demonstrates the increase in dark adaptation rate achieved with vector NVS2 generated by various production methods. NVS2 and NVS2a were both produced using two different CsCl gradient centrifugation methods while NVS2b was purified using column chromatography. Efficacy achieved 84 days post-injection with all three purification methods is indistinguishable based on a one way ANOVA with a Tukey's test. This result indicates that 3 independent productions of NVS2 in 2 independent laboratories yielded functional material resulting in similar efficacy in RLBP1 KO mice.


Summary of Example 4 Results:





    • Eyes injected with viral vector NVS2 exhibit an increased rate of dark adaption at doses ranging from 3×107 to 1×109 vg/eye, where efficacy lasts for at least 350 days post injection in the RLBP1 KO mouse model.

    • Eyes injected with viral vector NVS4 exhibit an increased rate of dark adaption at doses ranging from 3×108 to 1×109 vg/eye and the efficacy endures at least 204 days at both doses.

    • Eyes injected with viral vector NVS1 exhibit an increased rate of dark adaptation at the dose of 3×108 vg/eye and the efficacy endures at least 350 days.

    • Eyes injected with viral vector NVS3 and NVS5 exhibit an increased rate of dark adaptation at the dose of 3×109 vg/eye and efficacy endures at least 371 days. Efficacy of NVS3 and NVS5 was not observed at 3×108 vg/eye for any time point tested.





Conclusion:

Viral vector NVS2 exhibits higher maximum recovery than equivalent doses of the other vectors tested. Additionally, the NVS2 vector-mediated efficacy appears to be indistinguishable when prepared using CsCl or column chromatography purification.


Summary of Results:

The results demonstrated that self-complementary AAV8-pRLBP1(short)-eGFP vector, the reporter gene surrogate version of the therapeutic vector NVS2, leads to RPE and Müller cell type specific expression with no detectable off-target expression, where the therapeutic vector NVS2 leads to at least 350 days of visual function recovery measured by a-wave recovery in RLBP1 mice at doses ranging from 3×107 to 1×109 vg/eye. This specific gene cassette when packaged in a single-stranded genome and packaged with the same serotype capsid 8 exhibits significantly lower level of gene expression in mice, as demonstrated by the measurement of mRNA expression level. The same self-complementary genome as NVS2 and packaged in AAV2 capsid, which is NVS1, demonstrated efficacious a-wave recovery (i.e.: an increased rate of dark adaption) at the dose of 3×108 vg/eye for at least 350 days. This result suggests that NVS2 is a more potent viral vector than NVS1, which is likely due to the more efficient infection of AAV8 capsid than AAV2 capsid to the target cell types.


The results also demonstrated that AAV8-pRLBP1(long)-eGFP vector, the reporter gene surrogate version of the therapeutic vector NVS4, leads to RPE and Müller cell expression but also to photoreceptors. The therapeutic vector NVS4 leads to at least 204 days of efficacy at doses ranging from 3×108 to 1×109 vg/eye. The same genome in NVS4 but packaged in AAV 2 capsid, which is NVS3, leads to efficacious a-wave recovery at the dose of 3×109 but not at lower dose tested (3×108 vg/eye). The results demonstrated that AAV8-pRPE65-eGFP vector, the reporter gene surrogate version of the therapeutic vector NVS6, leads to RPE cell type expression with extensive photoreceptor off-target expression. When therapeutic vector NVS5, which carries the same genome as NVS6 but packaged into AAV2 capsid, is tested in RLBP1 KO mouse efficacy model, the results demonstrated that NVS5 endures positive a-wave recovery efficacy at the dose of 3×109 vg/eye but not at lower dose tested (3×108 vg/eye).


REFERENCES



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Claims
  • 1. A viral vector having a vector genome comprising a retinaldehyde binding protein 1 (RLBP1) coding sequence, wherein said vector is adapted to direct expression of an RLBP1 coding sequence in retinal pigment epithelial (RPE) cells and Müller cells of the retina.
  • 2. The viral vector of claim 1, wherein said vector comprises an adeno-associated virus (AAV) serotype 2 or 8 capsid.
  • 3. The viral vector of claim 2, said vector genome comprising in the 5′ to 3′ direction: (i) a 5′ ITR;(ii) a recombinant nucleotide sequence comprising an RLBP1 coding sequence; and(iii) a 3′ ITR.
  • 4. The viral vector of claim 3 where in the recombinant nucleotide sequence comprises in the 5′ to 3′ direction: (i) a promoter; (ii) an RLBP1 coding sequence; and (iii) an SV40 polyA sequence.
  • 5. The viral vector of 4, wherein the nucleotide sequence of said promoter is selected from SEQ ID NO: 3 and 10.
  • 6. The viral vector of claim 4 wherein the vector genome comprises in the 5′ to 3′ direction nucleic acid sequences selected from the group consisting of: a) SEQ ID NO: 2, 10, 5, 6, 8, and 9;b) SEQ ID NO: 2, 11, 5, 6, 8, 14, 9;c) SEQ ID NO: 2, 22, 5, 6, 8, 23, and 9; andd) SEQ ID NO: 2, 3, 4, 5, 6, 8, 23, and 9.
  • 7. The viral vector of claim 2, wherein said vector genome comprises in the 5′ to 3′ direction: (i) a 5′ ITR;(ii) a first recombinant nucleotide sequence;(iii) a non-resolvable ITR;(iv) a second recombinant nucleotide sequence; and(v) a 3′ ITR,wherein the first and second recombinant nucleotide sequences are self-complementary.
  • 8. The viral vector of claim 7 where in the second recombinant nucleotide sequence comprises in the 5′ to 3′ direction: (i) a promoter; (ii) an RLBP1 coding sequence; and (ii) an SV40 polyA sequence.
  • 9. The viral vector of 8, wherein the nucleotide sequence of said promoter comprises SEQ ID NO: 3.
  • 10. The viral vector of claim 8 wherein the vector genome comprises nucleic acid sequences in the 5′ to 3′ direction of: SEQ ID NO: 36, 62, 63, 64, 65, 66, 1, 3, 4, 5, 6, 8, and 9.
  • 11. A viral vector comprising a vector genome capable of expressing a heterologous gene in RPE cells and Müller cells of the retina, said vector comprising a) an AAV8 or AAV2 capsid and b) said vector genome comprising an RLBP1 promoter operably linked to the heterologous gene, wherein said RLBP1 promoter has a sequence selected from SEQ ID NOs: 3 and 10.
  • 12. The viral vector of claim 11, wherein the vector genome is self-complementary.
  • 13. A composition comprising the viral vector of any of the preceding claims.
  • 14. The composition of claim 13 further comprising a pharmaceutically acceptable excipient.
  • 15. A method of expressing a heterologous gene in retinal cells, the method comprising contacting said retinal cells with a viral vector comprising: a) an AAV2 or AAV8 capsid, and b) a vector genome comprising an RLBP1 promoter of SEQ ID NO: 3 or SEQ ID NO: 10 operably linked to the heterologous gene.
  • 16. The method of claim 15, wherein the retinal cells are RPE cells and Müller cells.
  • 17. The method of claim 16, wherein the vector genome is a self-complementary genome.
  • 18. The method of any of claims 15-17, wherein the heterologous gene is RLBP1.
  • 19. A nucleic acid comprising a gene cassette, said gene cassette comprises, in the 5′ to 3′ direction: (i) a 5′ ITR or a non-resolvable ITR;(ii) a recombinant nucleotide sequence comprising an RLBP1 coding sequence; and(iii) a 3′TR.
  • 20. The nucleic acid of claim 19 that is a plasmid.
  • 21. The nucleic acid of claim 19, wherein: (i) said 5′ ITR or said non-resolvable ITR have a nucleic acid sequence of SEQ ID NO: 2 or 1, respectively;(ii) said recombinant nucleic acid sequence comprises a promoter having a nucleic acid sequence selected from: SEQ ID NO: 3, 10, 11, 12 and 22 operably linked to said RLBP1 coding sequence having a nucleic acid sequence of SEQ ID NO: 3; or(ii) said 3′ ITR has a nucleic acid sequence of SEQ ID NO: 9.
  • 22. The nucleic acid of claim 19, wherein the gene cassette comprises a nucleic acid sequence selected from: SEQ ID NO: 51, 52, 53, 54, and 55.
  • 23. The nucleic acid of claim 19, further comprising the nucleic acid sequence selected from SEQ ID NO: 26, 27, 28, 29, 30 and 50.
  • 24. The nucleic acid of claim 19, wherein the gene cassette comprises, in the 5′ to 3′ direction, the sequences selected from: a) SEQ ID NO: 2, 10, 5, 6, 8, and 9,b) SEQ ID NO: 2, 11, 5, 6, 8, 14 and 9,c) SEQ ID NO: 2, 22, 5, 6, 8, 23 and 9,d) SEQ ID NO: 2, 3, 4, 5, 6, 8, 23 and 9, ore) SEQ ID NO: 1, 3, 4, 5, 6, 8, and 9.
  • 25. A method of treating a subject having RLBP1-associated retinal dystrophy, the method comprising administering to a subject the composition of claim 13.
  • 26. A method of improving the rate of dark adaption in a subject having RLBP1-associated retinal dystrophy, the method comprising administering to a subject the composition of claim 13.
  • 27. A method of expressing an RLBP1 coding sequence in RPE cells and Müller cells in the retina of a subject having RLBP1-associated retinal dystrophy, said method comprising the step of: contacting the retina of said subject with the viral vector of claim 6 or 10.
  • 28. The composition of claim 13 for use in treating a subject having RLBP1-associated retinal dystrophy.
  • 29. The composition of claim 13 for use in improving the rate of dark adaption in a subject having RLBP1-associated retinal dystrophy.
  • 30. The viral vector of claim 6 or 10 for use in expressing an RLBP1 coding sequence in RPE cells and Müller cells in the retina of a subject having RLBP1-associated retinal dystrophy.
RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/642,630 filed May 4, 2012 and U.S. Provisional Application No. 61/776,167 filed Mar. 11, 2013, the contents of which are incorporated herein by reference in their entireties.

Provisional Applications (2)
Number Date Country
61776167 Mar 2013 US
61642630 May 2012 US