Patent Application
20100184838
The invention relates to compositions and methods for delivery of gene products for treatment of ocular diseases.
A wide variety of eye diseases cause visual impairment, including macular degeneration, diabetic retinopathies, inherited retinal degeneration disorders such as retinitis pigmentosa, glaucoma, retinal detachment or injury and retinopathies (including those that are inherited, induced by surgery, trauma, a toxic compound or an agent, or induced photically).
A structure in the eye particularly affected by disease is the retina, found at the back of the eye, which is a specialized light-sensitive tissue that contains photoreceptor cells (rods and cones) and neurons connected to a neural network for the processing of visual information. The retina depends on cells of the adjacent retinal pigment epithelium (RPE) for support of its metabolic functions. Photoreceptors in the retina, perhaps because of their huge energy requirements and highly differentiated state, are sensitive to a variety of genetic and environmental insults. The retina is thus susceptible to an array of diseases that result in visual loss or complete blindness. Retinitis pigmentosa (RP), which results in the destruction of photoreceptor cells, the RPE, and the choroid, typifies inherited retinal degenerations. The RP group of debilitating conditions affects approximately 100,000 people in the United States. Compositions and methods are needed to treat RP and related diseases.
An embodiment of the invention provided herein is a method of treating an ocular condition in a subject, the method including administering intraocularly a recombinant adenovirus gene delivery vector comprising a eukaryotic promoter and a gene encoding a therapeutic protein, such that the promoter modulates expression of the gene and expressing the therapeutic protein treats the ocular condition. For example, the promoter originates from an eye of a vertebrate animal, for example the animal is of mammalian or avian origin, for example, the promoter originates from a gene expressed in a cell that is a rod or a cone of the eye of mammalian or avian origin. In various embodiments, the promoter is from a gene selected from at least one of group of a beta actin, a peripherin/RDS, cGMP phosphodiesterase, and a rhodopsin.
In various embodiments the gene encoding the therapeutic protein is at least one selected from the group of: an ATP binding casette retina gene (ABCR) gene, a glial cell derived neurotrophic factor (GDNF), a rhodopsin, a cyclic GMP phosophodiesterase, an alpha subunit of cyclic GMP phosophodiesterase (PDE6A), a beta subunit of cyclic GMP phosophodiesterase (PDE6B), an alpha subunit of rod cyclic nucleotide gated channel (CNGA1), a retinal pigmented epithelium-specific 65 kD protein gene (RPE65), a retinal binding protein 1 gene (RLBP1), a peripherin/retinal degeneration slow gene, a rod outer segment membrane protein 1 gene (ROM1), an arrestin (SAG), an alpha-transducin (GNAT1), a rhodopsin kinase (RHOK), a guanylate cyclase activator 1A (GUCA1A), a retina specific guanylate cyclase (GUCY2D), an alpha subunit of a cone cyclic nucleotide gated cation channel (CNGA3), and a cone opsin such as blue cone protein (BCP), green cone protein (GCP), and red cone protein (RCP). Exemplary genes encode a protein selected from a rhodopsin and a photoreceptor cell-specific ATP-binding transporter (ABCR).
In general in the methods herein, the adenovirus vector comprises a deletion in an adenovirus coat protein gene, the deletion encoding amino acid sequence arginine-glycine-aspartic acid (RGD domain).
In general, the step of administering is by a route selected from the group consisting of: administering in a contact lens fluid, contact lens cleaning and rinsing solutions, eye drops, surgical irrigation solutions, opthalmological devices, intravitreal injection, and subretinal injection. Exemplary methods of administering are by subretinal or intravitreal injection.
Another embodiment of the invention provided herein is a method of treating a subject for a condition of an eye, the method comprising administering intraocularly a recombinant adenovirus gene delivery vector wherein the vector nucleic acid comprises a first nucleotide sequence that encodes a modified coat protein, and a second nucleotide sequence that encodes a therapeutic protein, and expressing the second nucleotide sequence under the direction of a non-viral promoter. Thus the first nucleotide sequence further comprises a deletion encoding amino acid sequence arginine-glycine-aspartic acid (RGD domain). The promoter is for example from a gene selected from at least one of group of a beta actin, a peripherin/RDS, cGMP phosphodiesterase, and a rhodopsin. Exemplary non-viral promoters include a rhodopsin promoter or a beta actin promoter.
Further, the therapeutic protein is at least one selected from the group of: an ATP binding casette retina gene (ABCR) gene, a glial cell derived neurotrophic factor (GDNF), a rhodopsin, a cyclic GMP phosophodiesterase, an alpha subunit of cyclic GMP phosophodiesterase (PDE6A), a beta subunit of cyclic GMP phosophodiesterase (PDE6B), an alpha subunit of rod cyclic nucleotide gated channel (CNGA1), a retinal pigmented epithelium-specific 65 kD protein gene (RPE65), a retinal binding protein 1 gene (RLBP1), a peripherin/retinal degeneration slow gene, a rod outer segment membrane protein 1 gene (ROM1), an arrestin (SAG), an alpha-transducin (GNAT1), a rhodopsin kinase (RHOK), a guanylate cyclase activator 1A (GUCA1A), a retina specific guanylate cyclase (GUCY2D), an alpha subunit of a cone cyclic nucleotide gated cation channel (CNGA3), a cone opsin such as blue cone protein (BCP), green cone protein (GCP), and red cone protein (RCP).
The method in various embodiments is exemplified by administering by subretinal or intravitreal injection. Further, expressing the gene encoding the therapeutic protein includes expressing that gene in photoreceptor cells. In general, the adenovirus vector is a gutted vector.
Yet another embodiment of the invention provided herein is a method of treating or preventing macular degeneration in a subject diagnosed with or at risk for macular degeneration, the method comprising: administering to the subject a composition comprising a recombinant adenovirus gene delivery vector, the vector comprising a nucleotide sequence encoding: a modified coat protein, and a non-viral promoter operably linked to and directing expression of a gene that treats or prevents macular degeneration in the subject. In general, the modified coat protein has a deleted RGD domain.
Yet another embodiment of the invention provided herein is a method of treating or preventing retinitis pigmentosa in a subject diagnosed with or at risk for retinitis pigmentosa, the method including:
contacting the subject with a composition comprising a recombinant adenovirus gene delivery vector, the vector comprising nucleic acid encoding a modified coat protein and a therapeutic protein gene operably linked to a non-viral promoter, wherein the promoter directs expression of the therapeutic protein, and
administering intraocularly the composition to the subject, whereby the retinitis pigmentosa in the subject is treated or prevented. In general, the modified coat protein comprises a nucleotide sequence having a deletion of an RGD domain.
Yet another embodiment of the invention provided herein is a composition comprising a recombinant adenovirus gene delivery vector, the vector comprising a first nucleotide sequence encoding a modified viral coat protein and a second nucleotide sequence encoding a protein for expression in an ocular tissue, wherein the second nucleotide sequence is operably and regulatably linked to a non-viral promoter that directs expression of the second sequence. In general, the modified coat protein has a deleted RGD domain. Further, the promoter is of warm-blooded animal origin, for example, the promoter is of mammalian or avian origin, for example, the mammalian promoter is of human origin.
Yet another embodiment of the invention provided herein is a kit for preparing an adenoviral vector for delivery of a protein to ocular tissue, the kit comprising a nucleic acid encoding a viral coat protein deleted for amino acid sequence RGD and a eukaryotic promoter, and a container and instructions for recombinantly ligating a gene encoding a protein of interest.
Adenovirus (Ad) vectors have yielded substantial success in tests of rescue of retinal degeneration in animal models of human disease [1, 2]. In addition, two human ocular gene therapy trials have used Ad for gene delivery [3, 4]. These latter studies report that Ad is a useful vector for gene transfer to the human retina. Some advantages of Ad over other vector systems include a packaging capacity of 36 kb [5, 6], ease of scaled up production and episomal persistence and transgene expression extending over a period of years in non-human primates [7] or lifetime correction of disease in mice [8].
One of the most common diseases of the retina that leads to blindness is retinitis pigmentosa (RP), affecting approximately 1 in 3000 individuals worldwide [9]. A serotype of Ad used in gene therapy studies is Ad5, which infects the retinal pigment epithelium (RPE) upon subretinal administration [10-12]. Although the retinal pigment epithelium (RPE) provides essential components for the proper functioning and survival of the adjacent photoreceptor cells, the majority of genes associated with RP are expressed exclusively in the photoreceptors [13] and hence Ad has not heretofore been useful for the treatment of RP.
Other than pseudotyped adeno-associated virus (AAV), no previously described gene delivery vectors transduce photoreceptors efficiently in post mitotic retina. For example, while lentivirus vectors can transduce [14] and rescue [15] photoreceptor degeneration when administered to neonatal mice, these vectors were not found to transduce the fully developed photoreceptors in post mitotic adult murine retina [16-18]. Hence, lentivirus vectors have limited use in the treatment of diseases affecting human photoreceptors, that in contrast to murine photoreceptors are almost fully developed in utero [19]. Thus, it has been calculated that the stage most commonly used in retinal gene therapy experiments in mice (3 to 7 days post natal) corresponds to the second trimester in humans [20]. While AAV vectors can overcome this obstacle, they have a small cloning capacity of approximately 5 kb and hence cannot include the large upstream regulatory elements necessary for regulated expression of transgenes. Regulation of rhodopsin, for example, has been shown to be essential to prevent retinal degeneration and that induction of as little as 23% over-expression of wild type rhodopsin in photoreceptors may be sufficient [21]. Because of the above problems in current vector technology, development of adenovirus vectors specifically for retinal gene therapy is important.
An embodiment of the present invention provides a method of treating an eye of a subject, comprising administering intraocularly a recombinant adenovirus gene delivery vector, the vector comprising a non-viral promoter which directs the expression of a gene of interest. It is not intended that the present invention be limited to a particular non-viral promoter. The non-viral promoter is for example a ubiquitously expressed cellular promoter. An embodiment of the non-viral promoter is a chicken beta actin promoter (or portion thereof). The promoter confers cell specificity (e.g. wherein the non-viral promoter comprises a rhodopsin promoter). In one embodiment, the administering is by subretinal injection or by intravitreal injection.
In another embodiment the present invention provides a method of treating an eye of a subject, comprising administering intraocularly a recombinant adenovirus gene delivery vector, the vector comprising nucleic acid encoding a modified coat protein and a non-viral promoter which directs the expression of a gene of interest. In an embodiment, there is a deletion in the coding sequence for the coat protein (or portion thereof). In a particularly preferred embodiment, the modified coat protein has a deleted RGD domain. In an embodiment, the gene of interest is expressed predominantly (approximately 80% or more) in photoreceptor cells (and 20% or less in other cells of the eye). In a particularly preferred embodiment, the gene of interest is expressed almost exclusively (approximately 95% or more) in photoreceptor cells (and 5% or less in other cells of the eye).
In another embodiment the present invention provides a method of treating or preventing macular degeneration comprising: providing a subject diagnosed with or at risk for macular degeneration and a composition comprising a recombinant adenovirus gene delivery vector, the vector comprising nucleic acid encoding a modified coat protein and a non-viral promoter which directs the expression of a gene of interest, and intraocularly administering the composition to the subject. In an embodiment, the modified coat protein has a deleted RGD domain.
In yet another embodiment the present invention provides a method of treating or preventing retinitis pigmentosa comprising: providing a subject diagnosed with or at risk for retinitis pigmentosa and a composition comprising a recombinant adenovirus gene delivery vector, the vector comprising nucleic acid encoding a modified coat protein and a non-viral promoter which directs the expression of a gene of interest, and intraocularly administering the composition to the subject. In an embodiment, the modified coat protein has a deleted RGD domain.
An embodiment of the present invention provides a composition comprising a recombinant adenovirus gene delivery vector, the vector comprising nucleic acid encoding a modified coat protein and a non-viral promoter which directs the expression of a gene of interest. In an embodiment, the modified coat protein has a deleted RGD domain.
The adenoviral vector (in any of the above compositions or methods) is for example not replication competent (e.g. a so-called “gutted vector”). As used herein, the term “gutted viral vector” or “gutted viral DNA” refers to viral DNA that codes for viral vectors that contains cis-acting DNA sequences necessary for viral replication and packaging, but generally no viral coding sequences (U.S. Pat. No. 6,083,750, incorporated herein by reference). These vectors accommodate up to about 36 kb of exogenous DNA (heterogeneous DNA, i.e., DNA obtained from a different organism than the virus) and are unable to express viral proteins sufficient for replication. Helper-dependent viral vectors are produced by replication of the helper dependent viral DNA in the presence of a helper adenovirus, which alone or with a packaging cell line, supplies necessary viral proteins in trans such that the helper-dependent viral DNA is able to replicate (if necessary). Gutted vectors are constructed as described in U.S. Pat. No. 6,083,750.
As used herein, the term “gene of interest” or “transgene sequence” refers to a gene inserted into a vector or plasmid, expression of which (“expressing a protein of interest”) is desired in a host cell. Transgene sequences and transgene products include genes having therapeutic value as well as reporter genes. It is not intended that the present invention be limited by any particular gene of interest or mechanism of action or theory.
A variety of genes can be employed to treat eye disease, including but not limited to an ATP binding casette retina gene (ABCR) gene, a gene encoding a GDNF, and a rhodopsin gene (e.g. the opsin protein of rhodopsin). Other eye-specific therapeutic genes of interest include (but are not limited to) cyclic GMP phosophodiesterase (both the alpha subunit (PDE6A) and beta subunit (PDE6B)), the alpha subunit of the rod cyclic nucleotide gated channel (CNGA1), retinal pigmented epithelium-specific 65 kD protein gene (RPE65), retinal binding protein 1 gene (RLBP1), peripherin/retinal degeneration slow gene, rod outer segment membrane protein 1 gene (ROM1), and arrestin (SAG). These genes are exemplary for eye disease-associated genes, as they are all known to be mutated in retinitis pigmentosa (RP). In addition, other genes mutated in RP-related disorders include alpha-transducin (GNAT1), rhodopsin kinase (RHOK), guanylate cyclase activator 1A (GUCA1A), retina specific guanylate cyclase (GUCY2D), the alpha subunit of the cone cyclic nucleotide gated cation channel (CNGA3), and cone opsin genes such as blue cone protein gene (BCP), green cone protein gene (GCP), and red cone protein gene (RCP), which are mutated in certain forms of color blindness. In one embodiment, the adenoviral vectors of the present invention are employed to introduce “normal” or wild type, i.e., unmutated versions of such genes, in order to restore function or partial function in the eye.
An embodiment of the invention herein provides a method for in vivo gene therapy, by introducing an ABCR gene into targeted cells via intraocular injection (e.g. by subretinal or intravitreal routes of injection) of a nucleic acid construct or other appropriate delivery vectors, as shown in U.S. Pat. No. 6,713,300, hereby incorporated by reference. For example, a nucleic acid sequence encoding a ABCR protein product is recombinantly engineered in one of the adenovirus vectors described herein (and in particular, a gutted adenovirus) for delivery to the retinal cells. Such a method for therapy with ABCR is particularly useful for patients with Stargardt disease.
Another embodiment provides GDNF protein product for in vivo gene therapy by introducing a gene coding for GDNF protein into targeted cells via local injection of a nucleic acid construct or other appropriate delivery vectors, as shown in U.S. Pat. No. 5,736,516, hereby incorporated by reference. For example, a nucleic acid sequence encoding a GDNF protein product is engineered in one of the adenovirus vectors described herein for delivery to the retinal cells.
The present invention provides in one embodiment a method of utilizing the adenovirus vectors described herein to deliver ribozymes, such as those ribozymes shown in U.S. Pat. No. 6,225,291, hereby incorporated by reference.
Examples herein show the utility of Ad5 pseudotyped with Ad17 fiber (Ad5/F17), a serotype that has been previously shown to infect neurons in cell culture [22]. These examples show that Ad5/F17 vectors perform better than Ad5 vectors in ability to transduce neural retina, and not express high levels of transgene product in the photoreceptors.
Previous studies have concluded that the CMV promoter in the context of Ad5 in photoreceptor cells would be strongly active, and have attributed absence of reporter gene expression (GFP, LacZ etc.) in photoreceptors to be due to lack of photoreceptor transduction by Ad5. Hence these studies have reached the conclusion that Ad5 has an almost exclusive tropism for the RPE upon subretinal administration and that modifications such as pseudotyping are necessary in order to transduce photoreceptor cells.
In examples herein it is found on the contrary, that pseudotyping of Ad is in fact not necessary to achieve photoreceptor transduction. Rather, exchange of the transgene-associated promoter reveals that Ad5 in fact transduces photoreceptor cells to previously unprecedented levels.
These observations are expanded herein by probing the structural components of the Ad5 capsid for those that are associated with the substantially higher levels of RPE transduction at the expense of the photoreceptor cells. Given the robust integrin mediated phagocytic activity of the RPE for rod outer segment discs, studies herein were designed to test whether the RGD domain in Ad penton base plays a role in Ad5 uptake by the RPE. Examples herein demonstrate that deletion of the RGD domain in Ad penton base allows redirecting of Ad5 tropism from the much greater transduction of RPE cells to now more equivalent transduction of RPE and photoreceptors, at levels significantly greater than those achieved by pseudotyping or promoter exchange alone. These results were further developed to design adenovirus vectors that express transgenes strongly in the photoreceptor cells.
An aspect of the present invention provides pharmaceutical compositions, these compositions including the adenoviral vectors as described herein and in the claims, and optionally comprise a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent or agents are selected from the group consisting of growth factors, anti-inflammatory agents, vasopressor agents, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), and hyaluronic acid.
As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, surface active agents, isotonic agents, preservatives, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose, and sucrose; malt; gelatin; talc; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, and preservatives and antioxidants, according to the judgment of the formulator. Dosages of the vectors effective to treat or prevent ocular disease are described herein, and are to be adjusted to treat patients and subjects according to factors such as weight, age, and condition, as is known to one of ordinary skill in the art of pharmaceutical sciences.
A portion of this work appeared in an article entitled “Improved retinal transduction in vivo and photoreceptor-specific transgene expression using adenovirus vectors with modified penton base” by Siobhan M. Cashman, Laura McCullough and Rajendra Kumar-Singh, in Molecular Therapy 15:1640-1646, published September 2007, and which is hereby incorporated by reference herein in its entirety.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are within the scope of the present invention and claims. The contents of all references, including issued patents and published patent applications cited throughout this application, are hereby incorporated by reference.
pAd5/F17 and pShEGFPN were generated essentially as previously described for pAd5/F37 [35]. Wild-type Ad17 strain Ch.22, was obtained from the American Type Tissue Culture Collection. pShCAGGFP was constructed by cloning an Spell HindIII fragment of pCAGGFP into XbaI/HindIII-digested pShuttle [40]. pAdAdEasy1ΔRGD was constructed by cloning an FseI fragment from AdHM4ΔRGD into FseI-digested pAdEasy1 [40]. The 4.7 kb murine opsin promoter was cloned as a BamHI fragment from pSB6.25 and used to generate a GFP-expressing shuttle as described above for pShCAGGFP.
Viruses were produced as described previously [12, 40]. In brief, adenoviruses were produced by recombination between the respective shuttle plasmid and modified Ad backbone in BJ5183 cells. Recombined plasmids were digested with Pad, transfected into the human embryonic retinoblast (911) cell line [41], and resultant viruses purified using the Adenopure kit (Puresyn, Inc; Malvern Pa.).
Virus particles, 3.2×1010 were resuspended in 50 mMTris-HCl, pH 8.0/150 mM NaCl/0.1% SDS/1% Triton X-100 containing leupeptin (10 μg/ml), aprotinin (10 μg/ml), and PMSF (0.1 mM). Half of the particles were pre-incubated at 100° C. These were then loaded on a 12% denaturing gel (BMA, Rockland, Me.) and probed for fiber using the monoclonal antibody, Ab-4 (Clone 4D2, NeoMarkers) followed by an HRP-conjugated goat anti-mouse antibody (Jackson ImmunoResearch; West Grove Pa.).
C57Bl/6J mice were bred and maintained in a 12-hour light-dark cycle and cared for in accordance with federal, state and local regulations. The use of animals in this work was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were anesthetized by intraperitoneal injection of xylazine (10 mg/ml)/ketamine (1 mg/ml). Subretinal injections were performed using the transcleral approach with a 32G needle attached to a 5 μl glass syringe (Hamilton). 3×109 virus particles were injected into C57Bl/6J mice. 17.5 nMoles of the tetramethylrhodamine isothiocyanate-labeled 6-amino acid GRGDSP (SEQ ID NO: 4) peptide or GRGESP (SEQ ID NO: 5) peptide was injected into the sub-retinal space of C57Bl6/J mice. Eyes were harvested 90 mins post-injection and imaged as described below.
Retinas (n=4 per construct) were dissected and processed for total RNA using RNA STAT-60, according to manufacturer's instructions (Tel-Test, Inc, Friendswood, Tex.). DNAse-treated RNA was reverse-transcribed using oligod(T)16 and TaqMan (Applied Biosystems; Framingham Mass.) reverse transcription kit according to manufacturer's instructions. Single PCR reactions were performed on cDNA using TaqMan Universal PCR Master Mix (Applied Biosystems) and analyzed using the ABI PRISM 7900HT Sequence Detection System. GFP mRNA was detected using an assay custom designed by Applied Biosystems and the primer/probe combination
Normalization was performed using a TaqMan endogenous mouse β-actin control primer/probe combination (Applied Biosystems part no. 4352663). All probes contained the covalently-linked reporter FAM dye at their 5′ end and a TAMRA quencher dye at their 3′ end.
Six days post-injection (except for Ad5RhoGFPΔRGD, SEQ ID NO: 11, which was analyzed 3 weeks post-injection), mice were sacrificed by CO2 inhalation. Eyes were harvested and fixed with 4% paraformaldehyde prior to sectioning. Tissues were visualized with a Nikon Eclipse TS100 or Olympus IX51 microscope with a 120 W metal halide lamp and a GFP filter (excitation/emission maxima 474 nm/509 nm). Images were captured using Image Pro or CoolSnap software.
Previous studies have shown that Ad2 pseudotyped with Ad17 fiber can infect primary neuronal cells in culture [22]. Since the retina contains a variety of specialized neurons including photoreceptors, this example tests whether Ad5 pseudotyped with Ad17 fiber infects retinal neurons more efficiently than Ad5.
In order to construct such a vector, the fiber gene in Ad5 was genetically modified. In brief, modifications were made such that the resultant vector would express a hybrid fiber comprised of the N terminal 10 amino acids (aa) unique to Ad5 fiber followed by the amino acid sequence FNPVYPY (see for example SEQ ID NO: 1) that are common to both Ad5 and Ad17 fiber, followed by amino acids at positions 18 to 366 of Ad17, a total fiber length of 366 aa and identical in length to wild type Ad17 fiber (
To allow quantitation of potentially transduced neurons in vivo, a CMV-GFP expression cassette was cloned in the E1 region of this modified virus backbone (
The data obtained show that the monomeric fiber was expressed, trimerized and incorporated into virions and that denaturation by boiling samples prior to loading resolves the Ad5/F17 fibers into monomeric fibers (
To determine whether Ad5/F17 (EGFPNAd5/F17) could transduce retinal neurons, EGFPNAd5/F17 was injected into the subretinal space of adult C57BL/6J mice and GFP expression was measured qualitatively by direct fluorescence and quantitatively by qRT-PCR of neural retina (i.e. with RPE removed). Qualitatively, the data show that Ad5/F17 virions transduced the same cell types as the parental Ad5 virus, i.e. primarily RPE-specific GFP expression and the occasional Müller cell and cells of the inner nuclear layer were observed. However, qRT-PCR revealed that Ad5/F17 virus transduced murine neural retina 24±4-fold more efficiently than Ad5, the majority of the signal possibly due to slightly enhanced Müller cell transduction. However, Ad5/F17 did not transduce photoreceptors very effectively (
The assumption that the CMV promoter might be sufficient to determine which cell types are transduced by Ad in the retina has previously led investigators including the present inventors to conclude that photoreceptors are not transduced by Ad5 and alternative approaches such as pseudotyping might be necessary [23, 24].
To examine the role of the CMV promoter in Ad-transduced photoreceptors, Ad5 vectors expressing GFP were constructed from either a chicken beta actin promoter/CMV enhancer/rabbit globin intron [25] (Ad5CAGGFP; SEQ ID NO: 9) or a CMV promoter (Ad5CMVGFP) and were injected into the subretinal space of adult C57BL/6J mice. Examination of frozen retinal sections (
Closer examination of these retinal sections identified these GFP-positive cells as photoreceptor cell bodies (
In conclusion, these surprising data show that Ad5 vectors can transduce photoreceptor cells and that the CBA promoter allowed robust transgene expression in photoreceptors whereas the CMV promoter does not. Robust photoreceptor transduction was not limited to only the site of injection, and covered approximately 20% of the neural retina and approximately 35% of the RPE with a single subretinal injection (
Despite the observation herein that photoreceptors were in fact transduced by Ad5, the RPE was found to be substantially better transduced than the photoreceptors. To test whether the CBA promoter is equally active in both cell types, the molecular basis for the strong Ad-RPE interaction was investigated to determine potential enhancement of photoreceptor transduction by amelioration or reduction of such interaction. Since RPE is a phagocytic cell, whose function includes binding to and engulfing large amounts of rod outer segments (ROS) shed by the photoreceptor cells [26], these spent ROS might bind the RPE via an RGD-αvβ5 integrin interaction [27]. As Ad5 also binds αvβ5 integrin via an RGD domain in the Ad5 penton base [28] and this interaction is critical for Ad5 endocytosis following the initial Ad5 fiber knob-CAR receptor interaction [29], it was hypothesized herein that RGD-αvβ5-integrin interaction might be the basis for the very efficient uptake of Ad5 vectors into the RPE at the expense of Ad5 uptake by the adjacent photoreceptors.
To investigate this interaction further, a tetramethylrhodamine isothiocyanate-labeled ROD-containing peptide was injected into the subretinal space of adult C57BL/6J mice. Mouse eyes harvested 90 minutes later indicated that the ROD-containing peptide were found to bind primarily to the RPE and the blood vessels present in the inner retina (
If RGD were involved in the Ad-RPE interaction, an expected result would have been transduction primarily of the RPE and the blood vessels with RGD-containing Ad5 viruses expressing GFP from a promoter active in both these tissues. However, in examples herein, high levels of Ad5 transduction were observed only in the RPE and photoreceptors (
To further test the above interactions, a deletion of the RGD from the Ad5 vector was envisioned in order to reduce the Ad-RPE interaction and hence potentially make available more vector for uptake by adjacent cells such as the photoreceptors. To test this model, an Ad5 vector expressing GFP from a CBA promoter on an Ad5 backbone with an RGD deletion in Ad penton base (Ad5CAGGFPΔRGD; SEQ ID NO: 10) was designed, prepared and rescued. This construct was injected into the subretinal space of adult C57BL/6J mice and frozen retinal sections were examined six days later.
Several key differences were observed between Ad5CAGGFP (SEQ ID NO: 9) and Ad5CAGGFPΔRGD (SEQ ID NO: 10) injected mouse retina. The region of the neural retina that was transduced was substantially greater with the use of Ad5CAGGFPΔRGD (SEQ ID NO: 10), with as much as 50% of the neural retina transduced by a single injection (
Having demonstrated improved transgene expression in neural retina and specifically in photoreceptors using adenovirus vectors deleted in RGD-penton base, the vectors were examined for ability to express transgenes in a photoreceptor specific manner. As shown above, control of transgene expression is important for retinal gene therapy, and the 36 kb cloning capacity of helper dependent Ad vectors could be utilized to deliver for example the entire human rhodopsin gene including introns and large upstream regulatory elements or indeed other genes involved in retinal degeneration. Regulated photoreceptor specific transgene expression provides a further level of safety in human gene therapy protocols.
As a step towards that goal, an adenovirus vector (Ad5RhoGFPΔRGD; SEQ ID NO: 11) expressing GFP regulated by a 4.7 kb murine opsin promoter was generated herein. Frozen retinal sections prepared from mice three weeks after subretinal injection revealed GFP-expression in the area of subretinal injection (
Adenovirus vectors have had success in the two human ocular gene therapy trials to date. In both of these trials, a dose escalation study revealed no significant adverse events at the highest doses tested—109.5 [3] or 1011 [4] virion particle units per patient. Despite these reports of success, most ocular gene therapy studies in animals have not used Ad as the gene transfer vector [30]. The abandonment of Ad in animal ocular gene therapy has come about in part because of a substantial immune response observed in animals following administration of Ad into ocular tissues [31, 32]. However, in some murine ocular gene transfer studies it has been demonstrated that long term transgene expression is indeed possible, exceeding six months, the longest time periods examined [24, 33]. Given the promising data from the two human ocular gene therapy trial studies, exclusion of Ad in favor of alternative vectors may be unwarranted, especially given some of the advantages of Ad over other vector systems.
Examples herein examine the current perceived drawbacks of Ad vector technology for treatment of diseases involving photoreceptor degeneration. Similar to lentivirus, previous generations of Ad5 vectors can transduce photoreceptors only early during development [34]. Hence, while such vectors can be used to rescue degenerating photoreceptors in murine neonates [1, 2], they have not been considered to have much potential for application for similar diseases in humans. This is likely due to key differences in the timing of photoreceptor development between mice and humans after birth. This limitation in tropism is not valid for in utero gene transfer but technical and ethical hurdles are likely to slow such applications.
In order to address the above deficiencies, examples herein first examined the potential of photoreceptor transduction by Ad5 vectors pseudotyped with Ad17 fiber, a pseudotype known to infect post mitotic neuronal cells in culture [22]. Previous efforts to redirect Ad tropism from RPE to photoreceptors include Ad5 pseudotyped with Ad37 (Ad5/f37) [23] or Ad35 (Ad5/f35) [24] fiber. We have previously shown [35] that Ad5/f37 can be redirected from binding the native receptor to sialic acid, an amino carbohydrate abundantly present in the retina. In those former studies, Ad vectors expressing green fluorescent protein (GFP) from a CMV promoter were used to demonstrate photoreceptor transduction. GFP expression was observed directly in photoreceptors upon use of Ad5/f37 vector [23] but indirect and significantly more sensitive methods of detecting GFP, i.e. antibodies were used to detect GFP in photoreceptors with the Ad5/f35 vector [24]. Although those studies found an improvement in transduction of neural retina with Ad5/f17 vectors, it appeared to be due to slightly increased transduction of Müller cells and other cells in the inner nuclear layers but not in the photoreceptors.
Although the CMV promoter has been shown to be active in photoreceptors when CMV regulated expression cassettes have been delivered to photoreceptors by alternative vectors such as AAV [36], the reason for almost no transgene expression in photoreceptors in the context of Ad is surprising. One possible explanation for this observation is that Ad may generate a greater immune response than AAV in murine ocular tissues which may initiate a cascade of events that in part rapidly shut off viral promoters such as CMV. For example, IFNγ has been shown to bind and shut down expression from the CMV promoter as part of the natural host immune response [37]. Photoreceptor expression from CMV promoters in the context of Ad was tested herein with both sense and antisense orientations with respect to the E1 enhancer and similar results were obtained, implying that the location of the CMV promoter and its proximity to Ad E1 enhancers may not be relevant. Surprisingly, data in examples herein demonstrate substantial transduction of photoreceptors by use of the eukaryotic CBA promoter, implying that the obstacle in photoreceptor transduction is at least in part at the level of transcription, and not exclusively due to levels of infection as had been previously ascribed.
The role of RGD in penton base for Ad entry in non ocular cells in vivo has been examined previously. In those studies it was determined that RGD deletions did not substantially change the levels of Ad uptake, for example by liver following intravenous injection [38]. A variety of studies have enhanced Ad uptake into cells, often neoplastic cells that express high levels of integrin, by incorporation of RGD in the H1 loop of fiber knob [39]. Examples herein are hence atypical in that enhanced targeting of the cell of interest was achieved by reducing rather than increasing the RGD-integrin interaction. It was found that deletions in RGD surprisingly allowed substantial improvements in photoreceptor transduction.
Further examples herein show that a 4.7 kb murine opsin promoter was used to drive GFP expression exclusively in the photoreceptor cells. This is likely to be the largest photoreceptor-specific promoter delivered ectopically to photoreceptor cells thus far. While the deletion in Ad penton base (Ad5ΔRGD) allowed for improved photoreceptor transduction, use of the 4.7 kb opsin promoter on such viral backbones allowed expression exclusively in photoreceptor cells. Further without being limited by any particular theory or mechanism of action, the data herein support the idea that transgene levels from Ad5 vectors are likely to be lower than from Ad5ΔRGD vectors in photoreceptors due to an a priori difference in rates of photoreceptor transduction.
Vectors described in this study along with the further understanding of Ad tropism in ocular tissues will have applications in a variety of ocular diseases, without limitation, for example diseases involving the degeneration of the retina. Cumulatively, the data and vectors described in examples herein are useful for rescue of photoreceptors in animal models of retinal degeneration and in patients with retinitis pigmentosa and allied retinal disorders.
This application claims the benefit of U.S. provisional application 60/923,504 filed in the U.S. Patent and Trademark Office Apr. 13, 2007 entitled “Compositions and methods for improved retinal transduction and photoreceptor specific”, inventors R. Kumar-Singh and Siobhan M. Cashman, which is incorporated herein in its entirety by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/04744 | 4/11/2008 | WO | 00 | 10/13/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60923504 | Apr 2007 | US |
