OPTIMIZED PHENYLANANINE HYDROXYLASE EXPRESSION

FIELD

Aspects of the disclosure relate to genetic medicines for treating phenylketonuria (PKU). More specifically, aspects of the disclosure relate to lentiviral vectors, including codon-optimized PAH-containing lentiviral vectors.

BACKGROUND

Phenylketonuria (PKU) refers to a heterogeneous group of disorders that can lead to intellectual disability, seizures, behavioral problems, and impaired growth and development in affected children if left untreated. The mechanisms by which hyperphenylalaninemia results in intellectual impairment reflect the surprising toxicity of high dose phenylalanine and involve hypomyelination or demyelination of nervous system tissues. PKU has an average reported incidence rate of 1 in 12,000 in North America, affecting males and females equally. The disorder is most common in people of European or Native American ancestry and reaches much higher levels in the eastern Mediterranean region.

Neurological changes in patients with PKU have been demonstrated within one month of birth, and magnetic resonance imaging (MRI) in adult PKU patients has shown white matter lesions in the brain. The size and number of these lesions relate to blood phenylalanine concentrations. The cognitive profile of adolescents and adults with PKU compared with control subjects can include significantly reduced IQ, processing speed, motor control and inhibitory abilities, and reduced performance on tests of attention.

The majority of PKU is caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). PAH is a multimeric hepatic enzyme that catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine (Tyr) in the presence of molecular oxygen and catalytic amounts of tetrahydrobiopterin (BH₄), its nonprotein cofactor. In the absence of sufficient expression of PAH, phenylalanine levels in the blood increase leading to hyperphenylalaninemia and harmful side effects in PKU patients. Decreased or absent PAH activity can lead to a deficiency of tyrosine and its downstream products, including melanin, 1-thyroxine and the catecholamine neurotransmitters including dopamine.

PKU can be caused by mutations in PAH and/or a defect in the synthesis or regeneration of PAH cofactors (i.e., BH₄). Notably, several PAH mutations have been shown to affect protein folding in the endoplasmic reticulum resulting in accelerated degradation and/or aggregation due to missense mutations (63%) and small deletions (13%) in protein structure that attenuate or largely abolish enzyme catalytic activity.

In general, three major phenotypic groups are used to classify PKU based on blood plasma Phe levels, dietary tolerance to Phe and potential responsiveness to therapy. These groups include classical PKU (Phe >1200 μM), atypical or mild PKU (Phe is 600-1200 μM), and permanent mild hyperphenylalaninemia (HPA, Phe 120-600 μM).

Detection of PKU relies on universal newborn screening (NBS). A drop of blood collected from a heel stick is tested for phenylalanine levels in a screen that is mandatory in all 50 states of the USA.

Currently, lifelong dietary restriction of Phe and BH₄supplementation are the only two available treatment options for PKU, where early therapeutic intervention is critical to ensure optimal clinical outcomes in affected infants. However, costly medication and special low-protein foods impose a major burden on patients that can lead to malnutrition, psychosocial or neurocognitive complications notably when these products are not fully covered by private health insurance. Moreover, BH₄therapy is primarily effective for treatment of mild hyperphenylalaninemia as related to defects in BH₄biosynthesis, whereas only 20-30% of patients with mild or classical PKU are responsive. Thus, there is need for new treatment modalities for PKU as an alternative to burdensome Phe-restriction diets.

Genetic medicines have the potential to effectively treat PKU. Genetic medicines may involve delivery and expression of genetic constructs for the purposes of disease therapy or prevention. Expression of genetic constructs may be modulated by various promoters, enhancers, and/or combinations thereof.

SUMMARY

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a modified PAH sequence or variant thereof, for modulated phenylalanine hydroxylase (PAH) expression. In further aspects, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof, for enhanced PAH expression, and optionally a promoter and a liver-specific enhancer, wherein the PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In embodiments, the viral vector comprises a codon-optimized PAH sequence or variant thereof having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent sequence identity with SEQ ID NO: 70. In embodiments, the viral vector comprises a codon-optimized PAH sequence or variant thereof comprising the sequence of SEQ ID NO: 70.

In embodiments, the liver-specific enhancer comprises a prothrombin enhancer. In embodiments the prothrombin enhancer comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NO: 3. In embodiments, the prothrombin enhancer comprises the sequence of SEQ ID NO: 3.

In embodiments, the promoter comprises a liver-specific promoter. In embodiments, the liver-specific promoter comprises a hAAT promoter. In embodiments, the hAAT promoter comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NO: 4. In embodiments, the hAAT promoter comprises the sequence of SEQ ID NO: 4.

In embodiments, the therapeutic cargo portion further comprises a beta globin intron. In embodiments, the beta globin intron comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NOS: 5 or 6. In embodiments, the beta globin intron comprises the sequence of SEQ ID NOS: 5 or 6.

In embodiments, the therapeutic cargo portion further comprises at least one hepatocyte nuclear factor binding site. In embodiments, the hepatocyte nuclear factor binding site comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NOS: 7 (1XHNF1), 8 (5XHNF1), 9 (1XHNF1/4), or 10 (3XHNF1/4). In embodiments, the hepatocyte nuclear factor binding site comprises the sequence of SEQ ID NOS: 7, 8, 9, or 10.

In embodiments, the at least one hepatocyte nuclear factor binding site is disposed downstream of the prothrombin enhancer.

In embodiments, the therapeutic cargo portion further comprises at least one small RNA sequence. In embodiments, the at least one small RNA sequence comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NOS: 11 or 12. In embodiments, the at least one small RNA sequence is under the control of a first promoter and the PAH sequence is under the control of a second promoter. In embodiments, the first promoter is a H1 promoter. In embodiments, the second promoter is a liver-specific promoter.

In embodiments, the viral vector is a lentiviral vector or an adeno-associated viral vector. In embodiments, the viral vector is a lentiviral vector or another viral vector or non-viral system suitable for delivering the codon-optimized PAH sequence described herein. In embodiments, the viral vector is a lentiviral vector.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence that shares greater than 95 percent sequence identity to SEQ ID NO: 70. In embodiments, the codon-optimized PAH sequence or variant thereof comprises SEQ ID NO: 70.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion: wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 sequence identity to SEQ ID NO: 72. In embodiments, the codon-optimized sequence or variant thereof comprises SEQ ID NO: 72.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion: wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 73. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 73.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion: wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 74. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 74.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion: wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 76. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 76.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 73. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 73. In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises a codon-optimized PAH sequence or variant thereof, a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 74. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 74. In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, and further comprises a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 75. In embodiments, the codon-optimized sequence or variant thereof comprises SEQ ID NO: 75. In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, and further comprises a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 76. In embodiments, the codon-optimized sequence or variant thereof comprises SEQ ID NO: 76. In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, and further comprises a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In an aspect, a lentiviral particle produced by a packaging cell and capable of infecting a target cell is disclosed. In embodiments, the lentiviral particle comprises an envelope protein capable of infecting a target cell, and a viral vector as detailed herein.

In an aspect, a method of treating phenylketonuria (PKU) in a subject is disclosed. The method involves administering to the subject a therapeutically effective amount of a lentiviral particle as detailed herein.

In an aspect, use of a codon-optimized PAH sequence or variant thereof for treating PKU in a subject is provided. In another aspect, use of a codon-optimized PAH sequence or variant thereof to formulate a medicament for treating PKU in a subject is provided.

In an aspect, a codon-optimized PAH sequence or variant thereof for use in treating PKU in a subject is provided. In another aspect, a codon-optimized PAH sequence or variant thereof to formulate a medicament for use in treating PKU in a subject is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary 3-vector lentiviral vector system in a circularized form.

FIG. 2 depicts an exemplary 4-vector lentiviral vector system in a circularized form.

FIG. 3 depicts linear maps of four exemplary lentiviral vectors containing variations of the prothrombin enhancer and hAAT promoter to regulate the expression of PAH.

FIGS. 4A-4B depict immunoblot data comparing levels of PAH in Hepa1-6 cells after transduction of hPAH and various forms of codon-optimized PAH sequences. FIG. 4A compares hPAH with the OPT2 codon-optimized PAH. FIG. 4B compares hPAH with the OPT3, OPT2/3, and OPT3/2 versions of codon-optimized PAH.

FIG. 5 depicts PAH RNA expression in Hepa1-6 cells transduced with lentiviral vectors expression hPAH and codon-optimized versions of PAH.

FIGS. 6A-6B depict immunoblot data comparing levels of codon-optimized PAH with HNF1 and HNF1/4 binding sites upstream of the prothrombin enhancer. FIG. 6A depicts immunoblot data in Hepa1-6 cells. FIG. 6B depicts immunoblot data in Hep3B cells.

FIG. 7 depicts immunoblot data comparing levels of codon-optimized PAH with a regulatory sequence containing either prothrombin enhancer/hAAT promoter/Minute Virus of Mouse intron or hAAT enhancer/transthyretin promoter/Minute Virus of Mouse intron.

FIG. 8 depicts immunoblot data comparing levels of codon-optimized PAH with a regulatory sequence containing a mutant WPRE sequence or short WPRE (WPREs) sequence, or a PAH or albumin 3′ UTR sequence.

DETAILED DESCRIPTION
Overview of the Disclosure

This disclosure relates to therapeutic vectors and delivery of the same to cells. In an aspect, the therapeutic vector is a viral vector comprising a therapeutic cargo portion: wherein the therapeutic cargo portion comprises: a codon-optimized PAH sequence or variant thereof; a promoter; and a liver-specific enhancer, wherein the PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer. In embodiments, the vectors include codon-optimized PAH sequences or variants thereof, and/or a liver-specific enhancer. In embodiments, the vectors include a small RNA that regulates host (i.e., endogenous) PAH protein expression. In embodiments, the viral vector is a lentiviral vector.

Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the specification unless otherwise indicated. See, e.g.: Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Any enzymatic reactions or purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

As used herein, the singular forms “a”, “an” and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context indicates otherwise. Also, as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

All numerical designations, e.g., percent, pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which can include variation, for example (+) or (−) an increment of 0.1% or 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will include the value and up to plus or minus 10% of the value. The term “about” also includes the exact value “X” in addition to minor increments of “X” such as “X”+0.1% or X−0.1%.

As used herein, the term “administration of” or “administering” means providing any of the disclosed vectors, vector compositions, pharmaceutical compositions, or other active agents disclosed herein to a subject in need of treatment in a form that can be introduced into that individual's body in a therapeutically useful form and therapeutically effective amount. Methods of administering the disclosed vectors, vector compositions, or other active agents can be any of the methods disclosed herein.

As used herein, the phrase “coding sequence” describes any viral vector sequence capable of being transcribed or reverse transcribed. A “coding sequence” includes, without limitation, exogenous sequences (e.g., sequences on vectors that have been transduced or transfected into cells) capable of being transcribed or reverse transcribed.

As used herein, the term “codon-optimized” means modulating a coding sequence according to at least one of the following; (i) substituting naturally occurring codon sequences with alternative codons that preserve the amino acid sequence of the encoded protein but alter the composition and/or structure of the encoding RNA; (ii) modulating the guanosine cytosine content of the coding sequence relative to the naturally occurring guanosine cytosine content of the coding sequence; (iii) modulating the number of CpG sites of the coding sequence relative to the number of CpG sites in naturally occurring coding sequence; and (iv) substituting the naturally occurring codon sequences with alternative codons relative to (ii) the guanosine cytosine content and/or (iii) the number of CpG sites. Codon optimization may comprise adjustment of codons in the context of tRNA expression in specific tissues and/or may comprise methods for evading the action of natural, tissue-specific shRNA or miRNA.

As used herein, the term “comprising” means that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, means excluding other elements of any essential significance to the composition or method. “Consisting of” means excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).

As used herein, the term “CpG site,” refers to regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5′-3′ direction. CpG sites occur with high frequency in genomic regions called CpG islands (or CG islands). Cytosines in CpG dinucleotides can be methylated to form 5-methylcytosines. In mammals, 70% to 80% of CpG cytosines are methylated. Methylating the cytosine within a gene can change its expression.

As used here, the term “UTR” refers generally to an untranslated region of messenger RNA (mRNA) that remains after RNA splicing is completed. As used herein, “3′ UTR” refers to an untranslated region of mRNA that immediately follows the translation termination codon. The 3′UTR is not translated into a resulting protein.

As used herein, the term “adeno-associated viral vector,” refers to a synthetic delivery system which makes use of structural components of adeno-associated virus to deliver therapeutic DNA cargo into cells or tissues. The term “adeno-associated viral vector” may also be referred to herein as an “AAV vector”.

As used herein, the term “adeno-associated virus,” refers to a small virus that generates a mild immune response, is capable of depositing an extrachromosomal DNA copy of itself in a host cell, occasionally integrates a DNA copy into the host genome, and is relatively non-pathogenic. Adeno-associated virus includes numerous natural and synthetic serotypes, including but not limited to AAV2, as described herein.

As used herein, the term “AAV/DJ” (also referred to herein as “AAV-DJ”) is a serotype of an AAV vector engineered from different AAV serotypes, which mediates higher transduction and infectivity rates than wild type AAV serotypes.

As used herein, the term “AAV2” (also referred to herein as “AAV/2” or “AAV-2”) is a naturally occurring AAV serotype.

As used herein, the term “ApoE enhancer” refers to an Apolipoprotein E enhancer.

As used herein, the term “expression”, “expressed”, or “encodes” refers to the process by which polynucleotides are transcribed into mRNA or reverse transcribed into DNA and/or the process by which transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Expression may include splicing of the mRNA in a eukaryotic cell or other forms of post-transcriptional modification or post-translational modification.

As used herein, the term “genetic medicine” or “genetic medicines” refers generally to therapeutics and therapeutic strategies that focus on genetic targets to treat a clinical disease or manifestation. The term “genetic medicine” encompasses gene therapy and the like.

As used herein, the term “hAAT” refers to a hAAT promoter.

As used herein, the term “hepatocyte nuclear factors” refers to transcription factors that are predominantly expressed in the liver. Types of hepatocyte nuclear factors include, but are not limited to, hepatocyte nuclear factor 1, hepatocyte nuclear factor 2, hepatocyte nuclear factor 3, and hepatocyte nuclear factor 4.

As used herein, the term “HNF” refers to hepatocyte nuclear factor. Accordingly, HNF1 refers to hepatocyte nuclear factor 1, HNF2 refers to hepatocyte nuclear factor 2, HNF3 refers to hepatocyte nuclear factor 3, and HNF4 refers to hepatocyte nuclear factor 4.

As used herein, the term “HNF binding site,” refers to a region of DNA to which an HNF transcription factor can bind. Accordingly, a HNF1 binding site is a region of DNA to which HNF1 can bind, and a HNF4 binding site is a region of DNA to which HNF4 can bind.

As used herein, the term “human beta globin intron” refers to a nucleic acid segment within the human beta globin gene that is spliced out during RNA maturation, and does not code for a protein.

As used herein, the terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammal subject, e.g., murine, porcine, bovine, canine, feline, equine, nonhuman primate or human primate.

As used herein, the term “LV” refers generally to “lentivirus.” As a non-limiting example, reference to “LV-PAH” is reference to a lentivirus that contains a PAH sequence and expresses PAH. The PAH sequence may be a hPAH sequence or a codon-optimized PAH sequence.

As used herein, the term “LV-Pro-hAAT-PAH” refers to an LV vector comprising a prothrombin enhancer, a hAAT promoter, and a PAH sequence.

As used herein, the term “packaging cell line” refers to any cell line that can be used to express a lentiviral particle.

As used herein, the term “percent identity” or “percent sequence identity”, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the “percent identity” or “percent sequence identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

As used herein, the term “phenylalanine hydroxylase” may also be referred to herein as PA. The term phenylalanine hydroxylase includes nucleotide and peptide sequences of all wild type, variant, and codon-optimized PAH sequences, including fragments of PAH sequences. Without limitation, the term phenylalanine hydroxylase includes reference to SEQ ID NOS: 1, 2, and 70-76, and further includes variants having at least about 75% identity therewith.

As used herein, the term “hPAH” refers to a PAH sequence derived from a human or a human source, the codons of which have not been synthetically altered.

As used herein, the term “phenylketonuria”, which is also referred to herein as “PKU”, refers to the chronic deficiency of phenylalanine hydroxylase, as well as all symptoms related thereto including mild and classical forms of disease. Treatment of “phenylketonuria”, therefore, may relate to treatment for all or some of the symptoms associated with PKU.

As used herein, the term “prothrombin enhancer” is a region on the prothrombin gene that can be bound by proteins, which results in transcription of the prothrombin gene.

As used herein, the term “Pro” refers to a prothrombin enhancer.

As used herein, the term “rabbit beta globin intron” refers to a nucleic acid segment within the rabbit beta globin gene that is spliced out during RNA maturation, and does not code for a protein.

As used herein, the term “small RNA” refers to non-coding RNA that are generally about 200 nucleotides or less in length and possess a silencing or interference function. In other embodiments, the small RNA is about 175 nucleotides or less, about 150 nucleotides or less, about 125 nucleotides or less, about 100 nucleotides or less, or about 75 nucleotides or less in length. Such RNAs include microRNA (miRNA), small interfering RNA (siRNA), double stranded RNA (dsRNA), and short hairpin RNA (shRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). “Small RNA” of the disclosure should be capable of inhibiting or knocking-down gene expression of a target gene, generally through pathways that result in the degradation of the target gene mRNA or pathways that prevent translation of the target gene mRNA.

As used herein, the term “shPAH” refers to a small hairpin RNA that targets PAH.

As used herein, the term “SEQ ID NO” is synonymous with the term “Sequence ID No.”

As used herein, the term “thyroxin binding globulin,” is a transport protein responsible for carrying thyroid hormones in the bloodstream. As used herein, the abbreviation “TBG” refers to thyroxin binding globulin.

As used herein, the term “therapeutically effective amount” refers to a sufficient quantity of the active agents of the present disclosure, in a suitable composition, and in a suitable dosage form to treat or prevent the symptoms, progression, or onset of the complications seen in patients suffering from a given ailment, injury, disease, or condition. The therapeutically effective amount will vary depending on the state of the patient's condition or its severity, and the age, weight, etc., of the subject to be treated. A therapeutically effective amount can vary, depending on any of a number of factors, including, e.g., the route of administration, the condition of the subject, as well as other factors understood by those in the art.

As used herein, the term “therapeutic vector” includes, without limitation, reference to a lentiviral vector or an adeno-associated viral (AAV) vector. Additionally, as used herein with reference to the lentiviral vector system, the term “vector” is synonymous with the term “plasmid”. For example, the 3-vector and 4-vector systems, which include the 2-vector and 3-vector packaging systems, can also be referred to as 3-plasmid and 4-plasmid systems.

As used herein, the term “treatment” or “treating” generally refers to an intervention in an attempt to alter the natural course of the subject being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, suppressing, diminishing or inhibiting any direct or indirect pathological consequences of the disease, ameliorating or palliating the disease state, and causing remission or improved prognosis. A “treatment” is intended to target the disease state and combat it, i.e., ameliorate or prevent the disease state. The particular treatment thus will depend on the disease state to be targeted and the current or future state of medicinal therapies and therapeutic approaches. A treatment may have associated toxicities.

As used herein, the term “truncated” may also be referred to herein as “shortened” or “without”.

As used herein, the term “variant” refers to a nucleotide sequence that, when compared to a reference sequence, contains at least one of a single nucleotide polymorphism, a single nucleotide variation, a conversion, an inversion, a duplication, a deletion, or a substitution. A “variant” includes amino acid sequences that derive from “variant” nucleotide sequences, as well as post-transcriptional and post-translational modifications thereto.

As considered herein, optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

The nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules provided in the disclosure. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Description of Aspects and Embodiments

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof and a promoter.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof and an enhancer.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof, a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof and an enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by the enhancer. In embodiments, the enhancer is a liver-specific enhancer.

In embodiments, any of the promoters described herein are at least one of a tissue-specific promoter, a constitutive promoter, and a synthetic promoter.

In embodiments, the tissue-specific promoter is a liver-specific promoter. In embodiments, the liver-specific promoter is a hAAT promoter. In embodiments, the hAAT promoter comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent with SEQ ID NO: 4. For example, in embodiments, the hAAT promoter comprises a sequence that is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 4. In embodiments, the hAAT promoter comprises the sequence of SEQ ID NO: 4.

In embodiments, any of the liver-specific enhancers described herein are at least one of a naturally occurring enhancer and a synthetic enhancer.

In embodiments, the liver-specific enhancer is a prothrombin enhancer. In embodiments, the prothrombin enhancer comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NO: 3. For example, in embodiments, the prothrombin enhancer comprises a sequence that is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 3. In embodiments, the prothrombin enhancer comprises SEQ ID NO: 3.

In embodiments, the viral vector comprises an enhancer that is 5′ to a promoter. In embodiments, the viral vector comprises an enhancer that is 3′ to a promoter.

In embodiments, any of the codon-optimized PAH sequences or variants thereof are variants of a naturally occurring PAH sequence. In embodiments, any of the codon-optimized PAH sequences or variants thereof are variants of a synthetic PAH sequence.

In embodiments, the viral vector comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent sequence identity with SEQ ID NO: 70. For example, in embodiments, the codon-optimized PAH sequence is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 70. In embodiments, the viral vector comprises a codon-optimized PAH sequence or variant thereof comprising the sequence of SEQ ID NO: 70. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 70.

In embodiments, any of the therapeutic cargo portions described herein further comprises an intron. In embodiments, the intron is derived from any plant or animal species. In embodiments, the intron is a beta globin intron. In embodiments, the beta globin intron is a human beta globin intron. In embodiments, the beta globin intron is a rabbit beta globin intron. In embodiments, the beta globin intron comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NOS: 5 or 6. For example, in embodiments, the beta globin intron is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NOS: 5 or 6. In embodiments, the beta globin intron comprises the sequence of SEQ ID NOS: 5 or 6.

In embodiments, any of the therapeutic cargo portions described herein further comprise a site capable of being bound by a nuclear receptor. In embodiments, the nuclear receptor is expressed in the liver. In embodiments, the site is a hepatocyte nuclear factor binding site.

In embodiments, the hepatocyte nuclear factor binding site comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NOS: 7, 8, 9, or 10. For example, in embodiments, the hepatocyte nuclear factor binding site is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent 86 percent, 87 percent, 88 percent 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent identical to SEQ ID NOS: 7, 8, 9, or 10. In embodiments, the hepatocyte nuclear factor binding site comprises the sequence of SEQ ID NOS: 7, 8, 9, or 10.

In embodiments, any of the hepatocyte nuclear factor binding sites described herein are disposed downstream of a prothrombin enhancer. In embodiments, any of the hepatocyte nuclear factor binding sites described herein are disposed upstream of a prothrombin enhancer. As used herein, downstream refers to a distance measured in contiguous nucleotide positions along the direction of transcription for the functional RNA. Upstream refers to a distance measured in contiguous positions opposite to the direction of transcription for the functional RNA.

In embodiments, any of the therapeutic cargo portions described herein further comprise at least one small RNA sequence that is capable of binding to at least one pre-determined PAH mRNA sequence.

In embodiments, any of the at least one small RNA described herein is a small nuclear RNA. In embodiments, the at least one small RNA is a small nucleolar RNA. In embodiments, the at least one small RNA, is a microRNA. In embodiments, the at least one small RNA is a small interfering RNA. In embodiments, the at least one small RNA is a short hairpin RNA.

In embodiments, the at least one small RNA sequence comprises a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent identity with SEQ ID NOS: 11 or 12. For example, in embodiments, the at least one small RNA sequence is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NOS: 11 or 12. In embodiments, the at least one small RNA sequence comprises the sequence of SEQ ID NOS: 11 or 12.

In embodiments, any of the viral vectors described herein are at least one of a lentiviral vector and an AAV vector. In further embodiments, the following viral vectors can also be used in accordance with aspects of the present disclosure: Herpes simplex virus Type 1; Adenovirus, Moloney Murine Leukosis Virus; vectors based on oncoretroviruses including but not limited to HTLV-1 and HTLV-2; lentivirus vectors based on equine infectious anemia virus simian immunodeficiency virus, feline immunodeficiency virus, or Visna maedi lentivirus; measles virus vector; mumps virus vector; arbovirus vectors; equine infectious anemia virus vector; and vectors based on arenaviruses. In an aspect, gene delivery in accordance with the present disclosure may result in integration of a complementary gene copy at a location other than the gene encoding PAH, may result in creation of an extrachromosomal DNA or RNA element encoding PAH, may substitute for the natural PAH gene through homologous recombination, may utilize genome editing to insert a complementary gene sequence at or distant from the normal PAH gene or to exploit gene conversion to modify the sequence of chromosomal PAH genes. In another aspect, complementing DNA may be delivered in circular or linear forms through DNA transfection of liver, isolated hepatocytes or hepatocyte stem cells implanted into liver. In another aspect, complementing RNA may be delivered through transfection of liver, isolated hepatocytes or hepatocyte stem cells implanted into liver. In another aspect, isolated DNA or RNA may be delivered directly to accomplish gene conversion of the PAH gene, insert a complementing gene at a nearby or distant locus, or to modulate expression of negatively complementing chromosomal alleles of the PAH gene.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof, wherein the codon-optimized sequence or variant thereof having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 71. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 71. In embodiments, the codon-optimized sequence or variant thereof comprises the sequence of SEQ ID NO: 71. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 71.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and a liver-specific enhancer, wherein the codon-optimized sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and an enhancer.

In embodiments, the promoter can be any promoter described herein. In embodiments, the enhancer can be any enhancer described herein.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof, wherein the codon-optimized sequence or variant thereof having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 72. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 72. In embodiments, the codon-optimized sequence or variant thereof comprises the sequence of SEQ ID NO: 72. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 72.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and an enhancer.

In embodiments, the promoter can be any promoter described herein. In embodiments, the enhancer can be any enhancer described herein.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof, wherein the codon-optimized PAH sequence or variant thereof having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 73. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 73. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 73. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 73.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and an enhancer.

In embodiments, the promoter can be any promoter described herein. In embodiments, the enhancer can be any enhancer described herein.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof, wherein the codon-optimized PAH sequence or variant thereof having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 74. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 74. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 74. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 74.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and an enhancer.

In embodiments, the promoter can be any promoter described herein. In embodiments, the enhancer can be any enhancer described herein.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof, wherein the codon-optimized PAH sequence or variant thereof having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 75. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 75. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 75. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 75.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises a codon-optimized PAH sequence or variant thereof, a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and an enhancer.

In embodiments, the promoter can be any promoter described herein. In embodiments, the enhancer can be any enhancer described herein.

In an aspect, a viral vector is provided comprising a codon-optimized PAH sequence or variant thereof, wherein the codon-optimized PAH sequence or variant thereof having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 76. For example, in embodiments, the codon-optimized PAH sequence is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 76. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 76. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 73.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, a promoter, and an enhancer.

In embodiments, the promoter can be any promoter described herein. In embodiments, the enhancer can be any enhancer described herein.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence that shares greater than 90 percent sequence identity to SEQ ID NO: 70. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 70. In embodiments, the codon-optimized PAH sequence or variant thereof comprises SEQ ID NO: 70. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 70.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 sequence identity to SEQ ID NO 71. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 71. In embodiments, the codon-optimized PAH sequence or variant thereof comprises SEQ ID NO: 71. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 71.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 sequence identity to SEQ ID NO: 72. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 72. In embodiments the codon-optimized PAH sequence or variant thereof comprises SEQ ID NO: 72. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 72.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 73. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 73. In embodiments, the codon-optimized PAH sequence or variant thereof comprises SEQ ID NO: 73. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 73.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 74. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 74. In embodiments, the codon-optimized PAH sequence or variant thereof comprises SEQ ID NO: 74. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 74.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 75. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 75. In embodiments, the codon-optimized PAH sequence or variant thereof comprises the sequence of SEQ ID NO: 75. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 75.

In an aspect, a viral vector is provided comprising a therapeutic cargo portion, wherein the therapeutic cargo portion comprises a codon-optimized PAH sequence or variant thereof comprising a sequence having at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence identity to SEQ ID NO: 76. For example, in embodiments, the codon-optimized PAH sequence or variant thereof is 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent 97 percent, 98 percent, or 99 percent identical to SEQ ID NO: 76. In embodiments, the codon-optimized PAH sequence or variant thereof comprises SEQ ID NO: 76. In embodiments, the codon-optimized PAH sequence or variant thereof comprises a sequence having 90.0%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91.0%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.0%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94.0%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%. 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with SEQ ID NO: 76.

In embodiments, the viral vector further comprises a therapeutic cargo portion that comprises the codon-optimized PAH sequence or variant thereof, and further comprises a promoter, and a liver-specific enhancer, wherein the codon-optimized PAH sequence or variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.

In an aspect, a lentiviral vector is provided which enhances PAH sequence expression. In embodiments, at least one of a PAH sequence or PAH 3′UTR sequence is modified. In further embodiments, such modification alters the secondary structure of an mRNA transcript of the PAH sequence. In further embodiments, such modification comprises alteration of at least one of the mRNA PAH secondary structure sequence and the mRNA 3′ UTR secondary structure sequence. In further embodiments, such modification alters interactions of the coding region and 3′UTR region of PAH mRNA. In further embodiments, such modification inhibits the negative regulatory effects of PAH secondary structure on PAH protein production.

In embodiments, a modulated PAH sequence comprises any sequence in which the naturally occurring PAH sequence has been modified, including any addition, deletion, substitution, or modification of any one or more of its nucleotides, including any variants thereof. In embodiments, the modification comprises modulating one or more of the guanosine cytosine content of the naturally occurring sequence, one or more codons of the naturally occurring sequence, or one or more CpG sites of the naturally occurring sequence. In embodiments, the modification comprises a a codon-optimized PAH sequence. The PAH codon-optimized sequence may be any suitable PAH codon-optimized sequence, including those set forth and described herein. In embodiments, a vector that encodes a modified PAH sequence (including a codon-optimized sequence) results in higher PAH expression relative to a vector that encodes a PAH sequence that is not modified (e.g., that is not codon-optimized).

In embodiments, a modified PAH sequences comprises a sequence having at least 70%, 75%, 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity with any of SEQ ID NOs: 1, 70, 71 or 72. In embodiments the modified PAH comprises any of sequence of SEQ ID NOs: 70, 71 or 72.

In embodiments, a modulated PAH 3′UTR sequence comprises any sequence in which the naturally occurring PAH 3′ UTR sequence has been modified, including any addition, deletion, substitution, or modification of any one or more of its nucleotides, including any variants thereof. In embodiments, the modulated PAH 3′ UTR sequence comprises at least one of substitution or deletion of one or more of its nucleotides. In further embodiments all, or substantially all, of the 3′ UTR nucleotides are substituted or deleted.

In embodiments, the modified 3′UTR sequence comprises a 3′UTR sequence that is derived from a 3′UTR sequence of a different gene. In embodiments, the 3′UTR sequence of PAH is substituted with a 3′UTR sequence of a different gene. In embodiments, the 3′UTR sequence comprises albumin 3′UTR. In embodiments, the albumin 3′UTR comprises a sequence having at least 70%, 75%, 80%, at least 85%, at least 90%, or at least 95%, but less than 100%, sequence identity with SEQ ID NO: 86. In embodiments, the albumin 3′UTR comprises the sequence of SEQ ID NO: 86.

In embodiments, a lentiviral vector that encodes a PAH sequence that comprises a modified PAH 3′UTR sequence results in higher PAH expression than a lentiviral vector that encodes a PAH sequence in which the PAH 3′UTR is not disrupted.

In embodiments, a lentiviral vector that encodes a modified PAH 3′UTR and a modified PAH sequence (including a codon-optimized sequence) results in higher PAH expression relative to a vector that encodes any of PAH 3′UTR that is not modified and/or a PAH sequence that is not modified (e.g., that is not codon-optimized).

Phenylketonuria

PKU is believed to be caused by mutations of PAH and/or a defect in the synthesis or regeneration of PAH cofactors (i.e., BH₄). Notably, several PAH mutations have been shown to affect protein folding in the endoplasmic reticulum resulting in accelerated degradation and/or aggregation due to missense mutations (about 63%) and small deletions (about 13%) in protein structure that attenuates or largely abolishes enzyme catalytic activity. As there are numerous mutations that can affect the functionality of PAH, an effective therapeutic approach for treating PKU will need to address the aberrant PAH and a mode by which replacement PAH can be administered and/or generated.

In general, three major phenotypic groups are classified in PKU based on Phe levels measured at diagnosis, dietary tolerance to Phe and potential responsiveness to therapy. These groups include classical PKU (about Phe >1200 μM), atypical or mild PKU (Phe is about 600-1200 μM), and permanent mild hyperphenylalaninemia (HPA, Phe 120-600 μM).

Detection of PKU relies on universal newborn screening (NBS). A drop of blood collected from a heel stick is tested for phenylalanine levels in a screen that is mandatory in all 50 states of the USA and used routinely in most developed countries.

Genetic Medicines

Genetic medicine includes reference to viral vectors that are used to deliver genetic constructs to host cells for the purposes of disease therapy or prevention.

Genetic constructs can include, but are not limited to, functional genes or portions of genes to correct or complement existing defects, DNA sequences encoding regulatory proteins, DNA sequences encoding regulatory RNA molecules including antisense, short hairpin RNA, short homology RNA, long non-coding RNA, small interfering RNA or others, and decoy sequences encoding either RNA or proteins designed to compete for critical cellular factors to alter a disease state. In embodiments, genetic medicine involves delivering these therapeutic genetic constructs to target cells to provide treatment or alleviation of a particular disease.

By delivering a functional PAH gene to the liver in vivo, PAH activity may be reconstituted leading to normal clearance of Phe in the blood therefore eliminating the need for dietary restrictions or frequent enzyme replacement therapies. The effect of this therapeutic approach may be improved by the targeting of a shRNA against endogenous PAN. In an aspect of the disclosure, a functional PAH gene or a variant thereof can also be delivered in utero if a fetus has been identified as being at risk to a PKU genotype. In embodiments, the functional PAH gene or a variant thereof is a codon-optimized PAH gene. In embodiments, the diagnostic step can be carried out to determine whether the fetus is at risk for a PKU phenotype. If the diagnostic step determines that the fetus is at risk for a PKU phenotype, then the fetus can be treated with the genetic medicines detailed herein. Treatment can occur in utero or in vitro.

Lentiviral Vector System

A lentiviral virion (particle) in accordance with various aspects and embodiments herein is expressed by a vector system encoding the necessary viral proteins to produce a virion (viral particle). In various embodiments, one vector containing a nucleic acid sequence encoding the lentiviral Pol proteins is provided for reverse transcription and integration, operably linked to a promoter. In another embodiment, the Pol proteins are expressed by multiple vectors. In other embodiments, vectors containing a nucleic acid sequence encoding the lentiviral Gag proteins for forming a viral capsid, operably linked to a promoter, are provided. In embodiments, this gag nucleic acid sequence is on a separate vector than at least some of the pol nucleic acid sequence. In other embodiments, the gag nucleic acid sequence is on a separate vector from all the pol nucleic acid sequences that encode pol proteins.

Numerous modifications can be made to the vectors herein, which are used to create the particles to further minimize the chance of obtaining wild type revertants. These include, but are not limited to deletions of the U3 region of the LTR, tat deletions and matrix (MA) deletions. In embodiments, the gag, pol and env vector(s) do not contain nucleotides from the lentiviral genome that package lentiviral RNA, referred to as the lentiviral packaging sequence.

In embodiments, the vector(s) forming the particle do not contain a nucleic acid sequence from the lentiviral genome that expresses an envelope protein. In embodiments, a separate vector that contains a nucleic acid sequence encoding an envelope protein operably linked to a promoter is used. In embodiments, this separate vector encoding the envelop protein does not contain a lentiviral packaging sequence. In one embodiment the sequence encoding the envelope nucleic acid sequence encodes a lentiviral envelope protein.

In another embodiment the envelope protein is not from the lentivirus, but from a different virus. The resultant particle is referred to as a pseudotyped particle. By appropriate selection of envelopes one can “infect” virtually any cell. For example, one can use an env gene that encodes an envelope protein that targets an endocytic compartment. Examples of viruses from which such env genes and envelope proteins can derive include the influenza virus (e.g., the Influenza A virus, Influenza B virus, Influenza C virus, Influenza D virus, Isavirus, Quaranjavirus, and Thogotovirus), the Vesiculovirus (e.g., Indiana vesiculovirus), alpha viruses (e.g., the Semliki forest virus, Sindbis virus, Aura virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Getah virus, Highlands J virus, Trocara virus, Una Virus, Ndumu virus, and Middleburg virus, among others), arenaviruses (e.g., the lymphocytic choriomeningitis virus, Machupo virus, Junin virus and Lassa Fever virus), flaviviruses (e.g., the tick-borne encephalitis virus, Dengue virus, hepatitis C virus, GB virus, Apoi virus, Bagaza virus, Edge Hill virus, Jugra virus, Kadam virus, Dakar bat virus, Modoc virus, Powassan virus, Usutu virus, and Sal Vieja virus, among others), rhabdoviruses (e.g., vesicular stomatitis virus, rabies virus), paramyxoviruses (e.g., mumps or measles) and orthomyxoviruses (e.g., influenza virus).

Other envelope proteins that can preferably be used include those derived from endogenous retroviruses (e.g., feline endogenous retroviruses and baboon endogenous retroviruses) and closely related gammaretroviruses (e.g., the Moloney Leukemia Virus, MLV-E, MLV-A, Gibbon Ape Leukemia Virus, GALV, Feline leukemia virus, Koala retrovirus, Trager duck spleen necrosis virus, Viper retrovirus, Chick syncytial virus, Gardner-Arnstein feline sarcoma virus, and Porcine type-C oncovirus, among others). These gammaretroviruses can be used as sources of env genes and envelope proteins for targeting primary cells. The gammaretroviruses are particularly preferred where the host cell is a primary cell.

Envelope proteins can be selected to target a specific desired host cell. For example, targeting specific receptors such as a dopamine receptor can be used for brain delivery. Another target can be vascular endothelium. These cells can be targeted using an envelope protein derived from any virus in the Filoviridae family (e.g., Cuevaviruses, Dianloviruses, Ebolaviruses, and Marburgviruses). Species of Ebolaviruses include Tai Forest ebolavirus, Zaire ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus.

In addition, in embodiments, glycoproteins can undergo post-transcriptional modifications. For example, in an embodiment, the GP of Ebola, can be modified after translation to become the GP1 and GP2 glycoproteins. In another embodiment, one can use different lentiviral capsids with a pseudotyped envelope (e.g., FIV or SHIV [U.S. Pat. No. 5,654,195]). A SHIV pseudotyped vector can readily be used in animal models such as monkeys.

Lentiviral vector systems as provided herein typically include at least one helper plasmid comprising at least one of a gag, pol, or rev gene. Each of the gag, pol and rev genes may be provided on individual plasmids, or one or more genes may be provided together on the same plasmid. In one embodiment, the gag, pol, and rev genes are provided on the same plasmid (e.g., FIG. 1). In another embodiment, the gag and pol genes are provided on a first plasmid and the rev gene is provided on a second plasmid (e.g., FIG. 2). Accordingly, both 3-vector (e.g., FIG. 1) and 4-vector (e.g., FIG. 2) systems can be used to produce a lentivirus as described herein. In embodiments, the therapeutic vector, at least one envelope plasmid and at least one helper plasmid are transfected into a packaging cell, for example a packaging cell line. A non-limiting example of a packaging cell line is the 293T/17 HEK cell line. When the therapeutic vector, the envelope plasmid, and at least one helper plasmid are transfected into the packaging cell line, a lentiviral particle is ultimately produced. Lentiviral vector systems as provided herein typically include at least one helper plasmid comprising at least one of a gag, pol, or rev gene. Each of the gag, pol and rev genes may be provided on individual plasmids, or one or more genes may be provided together on the same plasmid. In one embodiment, the gag, pol, and rev genes are provided on the same plasmid (e.g., FIG. 1). In another embodiment, the gag and pol genes are provided on a first plasmid and the rev gene is provided on a second plasmid (e.g., FIG. 2). Accordingly, both 3-vector and 4-vector systems can be used to produce a lentivirus as described herein. In embodiments, the therapeutic vector, at least one envelope plasmid and at least one helper plasmid are transfected into a packaging cell, for example a packaging cell line. A non-limiting example of a packaging cell line is the 293T/17 HEK cell line. When the therapeutic vector, the envelope plasmid, and at least one helper plasmid are transfected into the packaging cell line, a lentiviral particle is ultimately produced.

In another aspect, a lentiviral vector system for expressing a lentiviral particle is disclosed. The system includes a lentiviral vector as described herein; an envelope plasmid for expressing an envelope protein optimized for infecting a cell; and at least one helper plasmid for expressing gag, pol, and rev genes, wherein when the lentiviral vector, the envelope plasmid, and the at least one helper plasmid are transfected into a packaging cell line, a lentiviral particle is produced by the packaging cell line, wherein the lentiviral particle is capable of inhibiting production of PAH.

In another aspect, the lentiviral vector, which is also referred to herein as a therapeutic vector, includes the following elements: hybrid 5′ long terminal repeat (Rous Sarcoma virus (RSV) promoter/5′ long terminal repeat (LTR)) (SEQ ID NOS: 13-14), Psi packaging signal (RNA packaging site) (SEQ ID NO: 15), Rev-response element (RRE) (SEQ ID NO: 16), central polypurine tract (cPPT) (polypurine tract) (SEQ ID NO: 17), human alpha-1 anti-trypsin promoter (hAAT) (SEQ ID NO: 4), Phenylalanine hydroxylase (PAH) (SEQ ID NOS: 1, 2, and 70-76), long Woodchuck Post-Transcriptional Regulatory Element (WPRE) sequence (SEQ ID NO: 18), and delta U3 3′ LTR (SEQ ID NO: 19). In embodiments, the lentiviral vector, which is also referred to herein as a therapeutic vector, includes the following elements: hybrid 5′ long terminal repeat (Rous Sarcoma virus (RSV) promoter/5′ long terminal repeat (LTR)) (SEQ ID NOS: 13-14), Psi packaging signal (RNA packaging site) (SEQ ID NO: 15), Rev-response element (RRE) (SEQ ID NO: 16), central polypurine tract (cPPT) (polypurine tract) (SEQ ID NO: 17), H1 promoter (SEQ ID NO: 20), PAH shRNA (SEQ ID NOS: 11 and 12), human alpha-1 anti-trypsin promoter (hAAT) (SEQ ID NO: 4), long Woodchuck Post-Transcriptional Regulatory Element (WPRE) sequence (SEQ ID NO: 18), and delta U3 3′ LTR (SEQ ID NO: 19). In embodiments, sequence variation, by way of substitution, deletion, addition, or mutation can be used to modify the sequences references herein.

In another aspect, a helper plasmid includes the following elements: CMV enhancer/chicken beta actin promoter (SEQ ID NO: 21); HIV component gag (SEQ ID NO: 22); HIV component pol (SEQ ID NO: 23); HIV Int (SEQ ID NO: 24); HIV RRE (SEQ ID NO: 25); and HIV Rev (SEQ ID NO: 26). In another aspect, the helper plasmid may be modified to include a first helper plasmid for expressing the gag gene (SEQ ID NO: 22) and pol gene (SEQ ID NO: 23), and a second and separate plasmid for expressing the rev gene (SEQ ID NO: 26). In embodiments, sequence variation, by way of substitution, deletion, addition, or mutation can be used to modify the sequences references herein.

In another aspect, an envelope plasmid includes the following elements: cytomegalovirus (CMV) promoter (SEQ ID NO: 27) and vesicular stomatitis virus G glycoprotein (VSV-G) (SEQ ID NO: 28). In embodiments, sequence variation, by way of substitution, deletion, addition, or mutation can be used to modify the sequences references herein.

In various aspects, the plasmids used for lentiviral packaging are modified by substitution, addition, subtraction or mutation of various elements without loss of vector function. For example, and without limitation, the following elements can replace similar elements in the plasmids that comprise the packaging system: Elongation Factor-1 alpha (EF-1 alpha) and ubiquitin C (UbC) promoters can replace the CMV or CAG promoter. SV40 poly A and bGH poly A can replace the rabbit beta globin poly A. In another aspect, the HIV sequences in the helper plasmid can be constructed from different HIV strains or clades. For example, the VSV-G glycoprotein can be substituted with membrane glycoproteins derived from gammaretroviruses (e.g., gibbon ape leukemia virus, GALV, murine leukemia virus 10A1, MLV, Koala retrovirus, Trager duck spleen necrosis virus, Viper retrovirus, Chick syncytial virus, Gardner-Arnstein feline sarcoma virus, and Porcine type-C oncovirus, among others), endogenous retroviruses (e.g., feline endogenous virus (RD114), human endogenous retrovirus such as HERV-W, and baboon endogenous retrovirus, BaEV, among others), Lyssavirus (e.g., Rabies virus, FUG), mammarenavirus (e.g., lymphocytic choriomeningitis virus, LCMV, Influenza viruses such as the Influenza A virus, Influenza A fowl plague virus, FPV, Influenza B virus, Influenza C virus, Influenza D virus, Isavirus, Quaranjavirus, and Thogotovirus), Alphavirus (e.g., Ross River alphavirus, RRV, or Ebola viruses, EboV, such as Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus).

Various lentiviral packaging systems can be acquired commercially (e.g., Lenti-vpak packaging kit from OriGene Technologies, Inc., Rockville, Md.), and can also be designed as described herein. Moreover, it is within the skill of a person ordinarily skilled in the relevant art to substitute or modify aspects of a lentiviral packaging system to improve any number of relevant factors, including the production efficiency of a lentiviral particle.

In another aspect, adeno-associated viral (AAV) vectors can also be used. In embodiments, the AAV vector is an AAV-DJ serotype. In embodiments, the AAV vector is any of serotypes 1-11. In embodiments, the AAV serotype is AAV-2. In embodiments, the AAV vector is a non-natural type engineered for optimal transduction of human hepatocytes.

AAV Vector Construction. In aspects of the disclosure, the PAH coding sequence (SEQ ID NOS: 1, 2, and 70-76) and the prothrombin enhancer (SEQ ID NO: 3) with hAAT promoter (SEQ ID NO: 4) are inserted into the pAAV plasmid (Cell Biolabs, San Diego, Calif.). The PAH coding sequence with flanking EcoRI and SalI restriction sites is synthesized by Eurofins Genomics (Louisville, Ky.). The pAAV plasmid and PAH sequence are digested with EcoRI and SalI enzyme and ligated together. Insertion of the PAH sequence is verified by sequencing. Next, the prothrombin enhancer and hAAT promoter are synthesized by Eurofins Genomics (Louisville, Ky.) with flanking MluI and EcoRI restriction sites. The pAAV plasmid containing the PAH coding sequence and the prothrombin enhancer/hAAT promoter sequence are digested with MluI and EcoRI enzymes and ligated together. Insertion of the prothrombin enhancer/hAAT promoter are verified by sequencing.

Further, a representative AAV plasmid system for expressing PAH may comprise an AAV Helper plasmid, an AAV plasmid, and an AAV Rev/Cap plasmid. The AAV Helper plasmid may contain a Left ITR (SEQ ID NO: 29), a Prothrombin enhancer (SEQ ID NO: 3), a human Anti alpha trypsin promoter (SEQ ID NO: 4), a PAH element (SEQ ID NOS: 1, 2 and 70-76), a PolyA element (SEQ ID NO: 30), and a Right ITR (SEQ ID NO: 31). The AAV plasmid may contain a suitable promoter element (SEQ ID NO: 21 or SEQ ID NO: 27), an E2A element (SEQ ID NO: 32), an E4 element (SEQ ID NO: 33), a viral associated (VA) RNA element (SEQ ID NO: 34), and a PolyA element (SEQ ID NO: 30). The AAV Rep/Cap plasmid may contain a suitable promoter element (SEQ ID NO: 21 or SEQ ID NO: 27), a Rep element (SEQ ID NO: 35; AAV2 Rep), a Cap element (SEQ ID NOS: 36 (AAV2 Cap), 37 (AAV8 Cap), or 38 (AAV DJ Cap)), and a PolyA element (SEQ ID NO: 30).

In embodiments, an AAV/DJ plasmid is provided comprising a prothrombin enhancer and a PAH sequence (AAV/DJ-Pro-PAH). In embodiments, the PAH sequence is any of the codon-optimized PAH sequences disclosed herein. In embodiments, an AAV/DJ plasmid is provided comprising a prothrombin enhancer, an intron, and a PAH sequence (AAV/DJ-Pro-Intron-PAH). In embodiments, the intron is a human beta globin intron. In embodiments, the intron is a rabbit beta globin intron. In embodiments, an AAV/DJ plasmid is provided comprising GFP (AAV/DJ-GFP).

In embodiments, an AAV2 plasmid is provided comprising a prothrombin enhancer and a PAH sequence (AAV2-Pro-PAH). In embodiments, the PAH sequence is any of the codon-optimized PAH sequences disclosed herein. In embodiments, an AAV2 plasmid is provided comprising a prothrombin enhancer, an intron, and a PAH sequence (AAV2-Pro-Intron-PAH). In embodiments, the intron is a human beta globin intron. In embodiments, the intron is a rabbit beta globin intron. In embodiments, an AAV2 is provided comprising GFP (AAV2-GFP).

In embodiments, any of the AAV vectors disclosed herein may contain a coding sequence that expresses a regulatory RNA. In embodiments, the regulatory RNA is a lncRNA. In embodiments, the regulatory RNA is a microRNA. In embodiments, the regulatory RNA is a piRNA. In embodiments, the regulatory RNA is a shRNA. In embodiments, the regulatory RNA is a small RNA sequence comprising a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more percent identity with SEQ ID NOS: 11 or 12.

Production of AAV particles. The AAV-PAH plasmid may be combined with the plasmids pAAV-RC2 (Cell Biolabs) and pHelper (Cell Biolabs). The pAAV-RC2 plasmid may contain the Rep and AAV-2 capsid genes and pHelper may contain the adenovirus E2A, E4, and VA genes. The AAV capsid may also comprise the AAV-8 (SEQ ID NO: 39) or AAV-DJ (SEQ ID NO: 40) sequences. To produce AAV particles, these plasmids may be transfected in the ratio 1:1:1 (pAAV-PAH: pAAV-RC2: pHelper) into 293T cells. For transfection of cells in 150 mm dishes (BD Falcon), 10 micrograms of each plasmid may be added together in 1 ml of DMEM. In another tube, 60 microliters of the transfection reagent PEI (1 microgram/ml) (Polysciences) may be added to 1 ml of DMEM. The two tubes may be mixed together and allowed to incubate for 15 minutes. Then the transfection mixture may be added to cells and the cells are collected after 3 days. The cells may be lysed by freeze/thaw lysis in dry ice/isopropanol. Benzonase nuclease (Sigma) may be added to the cell lysate for 30 minutes at 37 degrees Celsius. Cell debris may then be pelleted by centrifugation at 4 degrees Celsius for 15 minutes at 12,000 rpm. The supernatant may be collected and then added to target cells.

Dosage and Dosage Forms

The disclosed compositions can be used for treating PKU patients during various stages of the disease. The disclosed vector compositions allow for short, medium, or long-term expression of genes or sequences of interest and episomal maintenance of the disclosed vectors. Accordingly, dosing regimens may vary based upon the condition being treated and the method of administration.

In embodiments, vector compositions may be administered to a subject in need in varying doses. Specifically, a subject may be administered about ≥10⁶infectious doses (where 1 dose is needed on average to transduce 1 target cell). More specifically, a subject may be administered about ≥10⁷, about ≥10⁸, about ≥b 10⁹, about ≥10¹⁰, about ≥10¹¹, or about ≥10¹²infectious doses per kilogram of body weight, or any number of doses in-between these values. Upper limits of dosing will be determined for each disease indication, and will depend on toxicity/safety profiles for each individual product or product lot.

Additionally, vector compositions of the present disclosure may be administered periodically, such as once or twice a day, or any other suitable time period. For example, vector compositions may be administered to a subject in need once a week, once every other week, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every eighteen months, every two years, every thirty months, or every three years.

In embodiments, the disclosed vector compositions are administered as a pharmaceutical composition. In embodiments, the pharmaceutical composition can be formulated in a wide variety of dosage forms, including but not limited to nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical application. Each of the dosage forms can comprise various solubilizing agents, disintegrating agents, surfactants, fillers, thickeners, binders, diluents such as wetting agents or other pharmaceutically acceptable excipients. The pharmaceutical composition can also be formulated for injection, insufflation, infusion, or intradermal exposure. For instance, an injectable formulation may comprise the disclosed vectors in an aqueous or non-aqueous solution at a suitable pH and tonicity.

The disclosed vector compositions may be administered to a subject via direct injection into the liver with guided injection. In some embodiments, the vectors can be administered systemically via arterial or venous circulation. In some embodiments, the vector compositions can be administered via guided cannulation to tissues immediately surrounding liver including spleen or pancreas. In some embodiments, the vector compositions can be administered via guided cannulation or needle to kidney. In some embodiments, the vector compositions can be administered via guided cannulation or needle to specific regions of the brain including the substantia nigra. In some embodiments, the vector composition may be delivered by injection into the portal vein or portal sinus, and may be delivered by injection into the umbilical vein.

The disclosed vector compositions can be administered using any pharmaceutically acceptable method, such as intranasal, buccal, sublingual, oral, rectal, ocular, parenteral (intravenously, intradermally, intramuscularly, subcutaneously, intraperitoneally), pulmonary, intravaginal, locally administered, topically administered, topically administered after scarification, mucosally administered, via an aerosol, in semi-solid media such as agarose or gelatin, or via a buccal or nasal spray formulation.

Further, the disclosed vector compositions can be formulated into any pharmaceutically acceptable dosage form, such as a solid dosage form, tablet, pill, lozenge, capsule, liquid dispersion, gel, aerosol, pulmonary aerosol, nasal aerosol, ointment, cream, semi-solid dosage form, a solution, an emulsion, and a suspension. Further, the pharmaceutical composition may be a controlled release formulation, sustained release formulation, immediate release formulation, or any combination thereof. Further, the pharmaceutical composition may be a transdermal delivery system.

In embodiments, the pharmaceutical composition can be formulated in a solid dosage form for oral administration, and the solid dosage form can be powders, granules, capsules, tablets or pills. In embodiments, the solid dosage form can include one or more excipients such as calcium carbonate, starch, sucrose, lactose, microcrystalline cellulose or gelatin. In addition, the solid dosage form can include, in addition to the excipients, a lubricant such as talc or magnesium stearate. In some embodiments, the oral dosage form can be immediate release, or a modified release form. Modified release dosage forms include controlled or extended release, enteric release, and the like. The excipients used in the modified release dosage forms are commonly known to a person of ordinary skill in the art.

In embodiments, the pharmaceutical composition can be formulated as a sublingual or buccal dosage form. Such dosage forms comprise sublingual tablets or solution compositions that are administered under the tongue and buccal tablets that are placed between the cheek and gum.

In embodiments, the pharmaceutical composition can be formulated as a nasal dosage form. Such dosage forms of this disclosure comprise solution, suspension, and gel compositions for nasal delivery.

In embodiments, the pharmaceutical composition can be formulated in a liquid dosage form for oral administration, such as suspensions, emulsions or syrups. In embodiments, the liquid dosage form can include, in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as humectants, sweeteners, aromatics or preservatives. In embodiments, the composition can be formulated to be suitable for administration to a pediatric patient.

In embodiments, the pharmaceutical composition can be formulated in a dosage form for parenteral administration, such as sterile aqueous solutions, suspensions, emulsions, non-aqueous solutions or suppositories. In embodiments, the solutions or suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil or injectable esters such as ethyl oleate.

The dosage of the pharmaceutical composition can vary depending on the patient's weight, age, gender, administration time and mode, excretion rate, and the severity of disease.

In embodiments, the treatment of PKU is accomplished by guided direct injection of the disclosed vector constructs into liver, using needle, or intravascular cannulation. In embodiments, the vectors compositions are administered into the cerebrospinal fluid, blood or lymphatic circulation by venous or arterial cannulation or injection, intradermal delivery, intramuscular delivery or injection into a draining organ near the liver.

The following examples are given to illustrate aspects of the present invention. It should be understood, however, that the inventions are not to be limited to the specific conditions or details described in these examples. All printed publications referenced herein are specifically incorporated by reference.

EXAMPLES
Example 1. Development of a Lentiviral Vector System

A lentiviral vector system was developed as summarized in FIG. 1 (circularized form).

Lentiviral particles were produced in 293T/17 HEK cells (purchased from American Type Culture Collection, Manassas, Va.) following transfection with the therapeutic vector, the envelope plasmid, and the helper plasmid. The transfection of 293T/17 HEK cells, which produced functional viral particles, employed the reagent Poly(ethylenimine) (PEI) to increase the efficiency of plasmid DNA uptake. The plasmids and DNA were initially added separately in culture medium without serum in a ratio of 3:1 (mass ratio of PEI to DNA). After 2-3 days, cell medium was collected and lentiviral particles were purified by high-speed centrifugation and/or filtration followed by anion-exchange chromatography. The concentration of lentiviral particles can be expressed in terms of transducing units/ml (TU/ml). The determination of TU was accomplished by measuring HIV p24 levels in culture fluids (p24 protein is incorporated into lentiviral particles), measuring the number of viral DNA copies per transduced cell by quantitative PCR, or by infecting cells and using light (if the vectors encode luciferase or fluorescent protein markers).

A 3-vector system (i.e., which includes a 2-vector lentiviral packaging system) was designed for the production of lentiviral particles. A schematic of the 3-vector system is shown in FIG. 1. Briefly, and with reference to FIG. 1, the top-most vector is a helper plasmid, which, in this case, includes Rev. The vector appearing in the middle of FIG. 1 is the envelope plasmid. The bottom-most vector is the therapeutic vector, as described herein.

Referring to FIG. 1, the Helper plus Rev plasmid includes a CMV enhancer/chicken beta actin promoter (SEQ ID NO: 21); a chicken beta actin intron (SEQ ID NO: 39); a HIV Gag (SEQ ID NO: 22); a HIV Pol (SEQ ID NO: 23); a HIV Integrase (SEQ ID NO: 24); a HIV RRE (SEQ ID NO: 25); a HIV Rev (SEQ ID NO: 26); and a rabbit beta globin poly A (SEQ ID NO: 40).

The envelope plasmid includes a CMV promoter (SEQ ID NO: 27); a beta globin intron (SEQ ID NO: 5 or 6); a VSV-G envelope glycoprotein (SEQ ID NO: 28); and a rabbit beta globin poly A (SEQ ID NO: 40).

Synthesis of a 3-vector system, which includes a 2-vector lentiviral packaging system containing the Helper (plus Rev) and Envelope plasmids, is disclosed.

Materials and Methods:

Construction of the helper plasmid: The helper plasmid was constructed by initial PCR amplification of a DNA fragment from the pNL4-3 HIV plasmid (NIH Aids Reagent Program) containing Gag, Pol, and Integrase genes. Primers were designed to amplify the fragment with EcoRI and NotI restriction sites which could be used to insert at the same sites in the pCDNA3 plasmid (Invitrogen). The forward primer was (5′-TAAGCAGAATTCATGAATTTGCCAGGAAGAT-3′) (SEQ ID NO: 41) and reverse primer was (5′-CCATACAATGAATGGACACTAGGCGGCCGCACGAAT-3′) (SEQ ID NO: 42).

The sequence for the Gag, Pol, Integrase fragment was as follows:

(SEQ ID NO: 43)

GAATTCATGAATTTGCCAGGAAGATGGAAACCAAA

AATGATAGGGGGAATTGGAGGTTTTATCAAAGTAA

GACAGTATGATCAGATACTCATAGAAATCTGCGGA

CATAAAGCTATAGGTACAGTATTAGTAGGACCTAC

ACCTGTCAACATAATTGGAAGAAATCTGTTGACTC

AGATTGGCTGCACTTTAAATTTTCCCATTAGTCCT

ATTGAGACTGTACCAGTAAAATTAAAGCCAGGAAT

GGATGGCCCAAAAGTTAAACAATGGCCATTGACAG

AAGAAAAAATAAAAGCATTAGTAGAAATTTGTACA

GAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGG

GCCTGAAAATCCATACAATACTCCAGTATTTGCCA

TAAAGAAAAAAGACAGTACTAAATGGAGAAAATTA

GTAGATTTCAGAGAACTTAATAAGAGAACTCAAGA

TTTCTGGGAAGTTCAATTAGGAATACCACATCCTG

CAGGGTTAAAACAGAAAAAATCAGTAACAGTACTG

GATGTGGGCGATGCATATTTTTCAGTTCCCTTAGA

TAAAGACTTCAGGAAGTATACTGCATTTACCATAC

CTAGTATAAACAATGAGACACCAGGGATTAGATAT

CAGTACAATGTGCTTCCACAGGGATGGAAAGGATC

ACCAGCAATATTCCAGTGTAGCATGACAAAAATCT

TAGAGCCTTTTAGAAAACAAAATCCAGACATAGTC

ATCTATCAATACATGGATGATTTGTATGTAGGATC

TGACTTAGAAATAGGGCAGCATAGAACAAAAATAG

AGGAACTGAGACAACATCTGTTGAGGTGGGGATTT

ACCACACCAGACAAAAAACATCAGAAAGAACCTCC

ATTCCTTTGGATGGGTTATGAACTCCATCCTGATA

AATGGACAGTACAGCCTATAGTGCTGCCAGAAAAG

GACAGCTGGACTGTCAATGACATACAGAAATTAGT

GGGAAAATTGAATTGGGCAAGTCAGATTTATGCAG

GGATTAAAGTAAGGCAATTATGTAAACTTCTTAGG

GGAACCAAAGCACTAACAGAAGTAGTACCACTAAC

AGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGG

AGATTCTAAAAGAACCGGTACATGGAGTGTATTAT

GACCCATCAAAAGACTTAATAGCAGAAATACAGAA

GCAGGGGCAAGGCCAATGGACATATCAAATTTATC

AAGAGCCATTTAAAAATCTGAAAACAGGAAAGTAT

GCAAGAATGAAGGGTGCCCACACTAATGATGTGAA

ACAATTAACAGAGGCAGTACAAAAAATAGCCACAG

AAAGCATAGTAATATGGGGAAAGACTCCTAAATTT

AAATTACCCATACAAAAGGAAACATGGGAAGCATG

GTGGACAGAGTATTGGCAAGCCACCTGGATTCCTG

AGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAG

TTATGGTACCAGTTAGAGAAAGAACCCATAATAGG

AGCAGAAACTTTCTATGTAGATGGGGCAGCCAATA

GGGAAACTAAATTAGGAAAAGCAGGATATGTAACT

GACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGA

CACAACAAATCAGAAGACTGAGTTACAAGCAATTC

ATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAAC

ATAGTGACAGACTCACAATATGCATTGGGAATCAT

TCAAGCACAACCAGATAAGAGTGAATCAGAGTTAG

TCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAA

AAAGTCTACCTGGCATGGGTACCAGCACACAAAGG

AATTGGAGGAAATGAACAAGTAGATAAATTGGTCA

GTGCTGGAATCAGGAAAGTACTATTTTTAGATGGA

ATAGATAAGGCCCAAGAAGAACATGAGAAATATCA

CAGTAATTGGAGAGCAATGGCTAGTGATTTTAACC

TACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGC

TGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCA

TGGACAAGTAGACTGTAGCCCAGGAATATGGCAGC

TAGATTGTACACATTTAGAAGGAAAAGTTATCTTG

GTAGCAGTTCATGTAGCCAGTGGATATATAGAAGC

AGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAG

CATACTTCCTCTTAAAATTAGCAGGAAGATGGCCA

GTAAAAACAGTACATACAGACAATGGCAGCAATTT

CACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGG

CGGGGATCAAGCAGGAATTTGGCATTCCCTACAAT

CCCCAAAGTCAAGGAGTAATAGAATCTATGAATAA

AGAATTAAAGAAAATTATAGGACAGGTAAGAGATC

AGGCTGAACATCTTAAGACAGCAGTACAAATGGCA

GTATTCATCCACAATTTTAAAAGAAAAGGGGGGAT

TGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACA

TAATAGCAACAGACATACAAACTAAAGAATTACAA

AAACAAATTACAAAAATTCAAAATTTTCGGGTTTA

TTACAGGGACAGCAGAGATCCAGTTTGGAAAGGAC

CAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTA

GTAATACAAGATAATAGTGACATAAAAGTAGTGCC

AAGAAGAAAAGCAAAGATCATCAGGGATTATGGAA

AACAGATGGCAGGTGATGATTGTGTGGCAAGTAGA

CAGGATGAGGATTAA.

Next, a DNA fragment containing the RRE, Rev, and rabbit beta globin poly A sequence with XbaI and XmaI flanking restriction sites was synthesized by Eurofins Genomics. The DNA fragment was then inserted into the plasmid at the XbaI and XmaI restriction sites The DNA sequence was as follows:

(SEQ ID NO: 44)

TCTAGAATGGCAGGAAGAAGCGGAGACAGCGACGA

AGAGCTCATCAGAACAGTCAGACTCATCAAGCTTC

TCTATCAAAGCAACCCACCTCCCAATCCCGAGGGG

ACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTG

GAGAGAGAGACAGAGACAGATCCATTCGATTAGTG

AACGGATCCTTGGCACTTATCTGGGACGATCTGCG

GAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAG

ACTTACTCTTGATTGTAACGAGGATTGTGGAACTT

CTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTG

GTGGAATCTCCTACAATATTGGAGTCAGGAGCTAA

AGAATAGAGGAGCTTTGTTCCTTGGGTTCTTGGGA

GCAGCAGGAAGCACTATGGGCGCAGCGTCAATGAC

GCTGACGGTACAGGCCAGACAATTATTGTCTGGTA

TAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATT

GAGGCGCAACAGCATCTGTTGCAACTCACAGTCTG

GGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTG

TGGAAAGATACCTAAAGGATCAACAGCTCCTAGAT

CTTTTTCCCTCTGCCAAAAATTATGGGGACATCAT

GAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAA

GGAAATTTATTTTCATTGCAATAGTGTGTTGGAAT

TTTTTGTGTCTCTCACTCGGAAGGACATATGGGAG

GGCAAATCATTTAAAACATCAGAATGAGTATTTGG

TTTAGAGTTTGGCAACATATGCCATATGCTGGCTG

CCATGAACAAAGGTGGCTATAAAGAGGTCATCAGT

ATATGAAACAGCCCCCTGCTGTCCATTCCTTATTC

CATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTT

TATATTTTGTTTTGTGTTATTTTTTTCTTTAACAT

CCCTAAAATTTTCCTTACATGTTTTACTAGCCAGA

TTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGC

TGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGC

AGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTT

TCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC

ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC

TGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT

TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAA

ACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGT

CAGCAACCATAGTCCCGCCCCTAACTCCGCCCATC

CCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC

GCCCCATGGCTGACTAATTTTTTTTATTTATGCAG

AGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAG

AAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTT

TTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAA

TGGTTACAAATAAAGCAATAGCATCACAAATTTCA

CAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT

GGTTTGTCCAAACTCATCAATGTATCTTATCAGCG

GCCGCCCCGGG

Finally, the CMV promoter of pCDNA3.1 was replaced with the CAG promoter (CMV enhancer, chicken beta actin promoter plus a chicken beta actin intron sequence). A DNA fragment containing the CAG enhancer/promoter/intron sequence with MluI and EcoRI flanking restriction sites was synthesized by Eurofins Genomics. The DNA fragment was then inserted into the plasmid at the MluI and EcoRI restriction sites. The DNA sequence was as follows:

(SEQ ID NO: 45)

ACGCGTTAGTTATTAATAGTAATCAATTACGGGGT

CATTAGTTCATAGCCCATATATGGAGTTCCGCGTT

ACATAACTTACGGTAAATGGCCCGCCTGGCTGACC

GCCCAACGACCCCCGCCCATTGACGTCAATAATGA

CGTATGTTCCCATAGTAACGCCAATAGGGACTTTC

CATTGACGTCAATGGGTGGACTATTTACGGTAAAC

TGCCCACTTGGCAGTACATCAAGTGTATCATATGC

CAAGTACGCCCCCTATTGACGTCAATGACGGTAAA

TGGCCCGCCTGGCATTATGCCCAGTACATGACCTT

ATGGGACTTTCCTACTTGGCAGTACATCTACGTAT

TAGTCATCGCTATTACCATGGGTCGAGGTGAGCCC

CACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCT

CCCCACCCCCAATTTTGTATTTATTTATTTTTTAA

TTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGG

CGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC

CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTA

TGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAG

CGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCC

TTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCC

GCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCA

CAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGG

CTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTT

CTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTC

CGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGG

GGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC

GCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGC

TGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGT

GTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCC

GCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTG

CGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGG

GTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCT

GCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGG

CTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG

GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGT

GGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCG

GGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGA

GCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGC

CATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCA

GGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAA

ATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGC

GCGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAA

TGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGC

CGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCG

CAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAG

GGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGGA

ATTC

Construction of the VSV-Envelope Plasmid:

The vesicular stomatitis Indiana virus glycoprotein (VSV-G) sequence was synthesized by Eurofins Genomics with flanking EcoRI restriction sites. The DNA fragment was then inserted into the pCDNA3.1 plasmid (Invitrogen) at the EcoRI restriction site and the correct orientation was determined by sequencing using a CMV specific primer.

The DNA sequence was as follows:

(SEQ ID NO: 28)

ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCAT

TGGGGTGAATTGCAAGTTCACCATAGTTTTTCCAC

ACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCT

AATTACCATTATTGCCCGTCAAGCTCAGATTTAAA

TTGGCATAATGACTTAATAGGCACAGCCTTACAAG

TCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCA

GACGGTTGGATGTGTCATGCTTCCAAATGGGTCAC

TACTTGTGATTTCCGCTGGTATGGACCGAAGTATA

TAACACATTCCATCCGATCCTTCACTCCATCTGTA

GAACAATGCAAGGAAAGCATTGAACAAACGAAACA

AGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAA

GTTGTGGATATGCAACTGTGACGGATGCCGAAGCA

GTGATTGTCCAGGTGACTCCTCACCATGTGCTGGT

TGATGAATACACAGGAGAATGGGTTGATTCACAGT

TCATCAACGGAAAATGCAGCAATTACATATGCCCC

ACTGTCCATAACTCTACAACCTGGCATTCTGACTA

TAAGGTCAAAGGGCTATGTGATTCTAACCTCATTT

CCATGGACATCACCTTCTTCTCAGAGGACGGAGAG

CTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAG

AAGTAACTACTTTGCTTATGAAACTGGAGGCAAGG

CCTGCAAAATGCAATACTGCAAGCATTGGGGAGTC

AGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGA

TAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAAT

GCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAG

ACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGA

GAGGATCTTGGATTATTCCCTCTGCCAAGAAACCT

GGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCA

GTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGG

AACCGGTCCTGCTTTCACCATAATCAATGGTACCC

TAAAATACTTTGAGACCAGATACATCAGAGTCGAT

ATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAAT

GATCAGTGGAACTACCACAGAAAGGGAACTGTGGG

ATGACTGGGCACCATATGAAGACGTGGAAATTGGA

CCCAATGGAGTTCTGAGGACCAGTTCAGGATATAA

GTTTCCTTTATACATGATTGGACATGGTATGTTGG

ACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTG

TTCGAACATCCTCACATTCAAGACGCTGCTTCGCA

ACTTCCTGATGATGAGAGTTTATTTTTTGGTGATA

CTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAA

GGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTC

TTTTTTCTTTATCATAGGGTTAATCATTGGACTAT

TCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATT

AAATTAAAGCACACCAAGAAAAGACAGATTTATAC

AGACATAGAGATGAACCGACTTGGAAAGTGA

A 4-vector system, which includes a 3-vector lentiviral packaging system, has also been designed and produced using the methods and materials described herein. A schematic of the 4-vector system is shown in FIG. 2. Briefly, and with reference to FIG. 2, the top-most vector is a helper plasmid, which, in this case, does not include Rev. The second vector is a separate Rev plasmid. The third vector is the envelope plasmid. The bottom-most vector is the therapeutic vector as described herein.

Referring to FIG. 2, the Helper plasmid includes a CMV enhancer/chicken beta actin promoter (SEQ ID NO: 21); a chicken beta actin intron (SEQ ID NO: 39); a HIV Gag (SEQ ID NO: 22); a HIV Pol (SEQ ID NO: 23); a HIV Integrase (SEQ ID NO: 24); a HIV RRE (SEQ ID NO: 25); and a rabbit beta globin poly A (SEQ ID NO: 40).

The Rev plasmid includes a RSV promoter and HIV Rev (SEQ ID NO: 46); and a rabbit beta globin poly A (SEQ ID NO: 40).

The Envelope plasmid includes a CMV promoter (SEQ ID NO: 27); a beta globin intron (SEQ ID NO: 5 or 6); a VSV-G envelope glycoprotein (SEQ ID NO: 28); and a rabbit beta globin poly A (SEQ ID NO: 40).

In one aspect, the therapeutic lentiviral vector expressing PAH includes all of the elements shown in Vector A of FIG. 3. In another aspect, the therapeutic lentiviral vector expressing PAH includes all of the elements shown in Vector B of FIG. 3. In another aspect, the therapeutic lentiviral vector expressing PAH includes all of the elements shown in Vector C of FIG. 3. In another aspect, the therapeutic lentiviral vector expressing PAH includes all of the elements shown in Vector D of FIG. 3.

Synthesis of a 4-vector system, which includes a 3-vector lentiviral packaging system containing the Helper, Rev, and Envelope plasmids, is disclosed.

Materials and Methods:

Construction of the Helper Plasmid without Rev:

The Helper plasmid without Rev was constructed by inserting a DNA fragment containing the RRE and rabbit beta globin poly A sequence. This sequence was synthesized by Eurofins Genomics with flanking XbaI and XmaI restriction sites. The RRE/rabbit poly A beta globin sequence was then inserted into the Helper plasmid at the XbaI and XmaI restriction sites.

The DNA sequence is as follows:

Construction of the Rev Plasmid:

The RSV promoter and HIV Rev sequences were synthesized as a single DNA fragment by Eurofins Genomics with flanking MfeI and XbaI restriction sites. The DNA fragment was then inserted into the pCDNA3.1 plasmid (Invitrogen) at the MfeI and XbaI restriction sites in which the CMV promoter is replaced with the RSV promoter. The DNA sequence was as follows:

(SEQ ID NO: 46)

CAATTGCGATGTACGGGCCAGATATACGCGTATCT

GAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGG

CTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGAT

ATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAAT

GTAGTCTTATGCAATACACTTGTAGTCTTGCAACA

TGGTAACGATGAGTTAGCAACATGCCTTACAAGGA

GAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGT

AAGGTGGTACGATCGTGCCTTATTAGGAAGGCAAC

AGACAGGTCTGACATGGATTGGACGAACCACTGAA

TTCCGCATTGCAGAGATAATTGTATTTAAGTGCCT

AGCTCGATACAATAAACGCCATTTGACCATTCACC

ACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTA

GTGAACCGTCAGATCGCCTGGAGACGCCATCCACG

CTGTTTTGACCTCCATAGAAGACACCGGGACCGAT

CCAGCCTCCCCTCGAAGCTAGCGATTAGGCATCTC

CTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAA

CTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTA

TCAAAGCAACCCACCTCCCAATCCCGAGGGGACCC

GACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGA

GAGAGACAGAGACAGATCCATTCGATTAGTGAACG

GATCCTTAGCACTTATCTGGGACGATCTGCGGAGC

CTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTT

ACTCTTGATTGTAACGAGGATTGTGGAACTTCTGG

GACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGG

AATCTCCTACAATATTGGAGTCAGGAGCTAAAGAA

TAGTCTAGA

The plasmids used in the packaging systems can be modified with similar elements, and the intron sequences can potentially be removed without loss of vector function. For example, the following elements can replace similar elements in the packaging system:

Promoters: Elongation Factor-1 alpha (EF1-alpha) promoter (SEQ ID NO: 47), phosphoglycerate kinase (PGK) promoter (SEQ ID NO: 48), thyroxin binding globulin promoter (SEQ ID NO: 60), and ubiquitin C (UbC) promoter (SEQ ID NO: 49) can replace the CMV promoter (SEQ ID NO: 27) or CMV enhancer/chicken beta actin promoter (SEQ ID NO: 21). These sequences can also be further varied by addition, substitution, deletion or mutation.

Poly A sequences: SV40 poly A (SEQ ID NO: 50) and bGH poly A (SEQ ID NO: 30 or SEQ ID NO: 51) can replace the rabbit beta globin poly A (SEQ ID NO: 40). These sequences can also be further varied by addition, substitution, deletion or mutation.

HIV Gag, Pol, and Integrase sequences: The HIV sequences in the Helper plasmid can be constructed from different HIV strains or clades. For example, HIV Gag (SEQ ID NO: 22); HIV Pol (SEQ ID NO: 23); and HIV Int (SEQ ID NO: 24) from the Bal strain can be interchanged with the gag, pol, and int sequences contained in the helper/helper plus Rev plasmids as outlined herein. These sequences can also be further varied by addition, substitution, deletion or mutation.

Envelope: The VSV-G glycoprotein can be substituted with membrane glycoproteins from feline endogenous virus (RD114) envelope (SEQ ID NO: 52), gibbon ape leukemia virus (GALV) envelope (SEQ ID NO: 53), Rabies (FUG) envelope (SEQ ID NO: 54), lymphocytic choriomeningitis virus (LCMV) envelope (SEQ ID NO: 55), influenza A fowl plague virus (FPV) envelope (SEQ ID NO: 56), Ross River alphavirus (RRV) envelope (SEQ ID NO: 57), murine leukemia virus 10A1 (MLV 10A1) envelope (SEQ ID NO: 58), or Ebola virus (EboV) envelope (SEQ ID NO: 59). Sequences for these envelopes are identified in the sequence portion herein. Further, these sequences can also be further varied by addition, substitution, deletion or mutation.

In summary, the 3-vector versus 4-vector systems can be compared and contrasted as follows. The 3-vector lentiviral vector system may comprise: (1) Helper plasmid: HIV Gag, Pol, Integrase fragment (SEQ ID NO: 43), RRE, and Rev; (2) Envelope plasmid: VSV-G envelope; and (3) Therapeutic vector: RSV, 5′LTR, Psi Packaging Signal, RRE, cPPT, prothrombin enhancer, alpha 1 anti-trypsin promoter, phenylalanine hydroxylase, WPRE, and 3′delta LTR. The 4-vector lentiviral vector system may comprise: (1) Helper plasmid: HIV Gag, Pol, Integrase fragment (SEQ ID NO: 43), and RRE; (2) Rev plasmid: Rev; (3) Envelope plasmid: VSV-G envelope; and (4) Therapeutic vector: RSV, 5′LTR, Psi Packaging Signal, RRE, cPPT, prothrombin enhancer, alpha 1 anti-trypsin promoter, phenylalanine hydroxylase, WPRE, and 3′delta LTR. Sequences corresponding with the above elements are identified in the sequence listings portion herein.

Example 2. Therapeutic Vectors

Exemplary therapeutic vectors have been designed and developed as shown, for example, in FIG. 3.

Referring first to Vector A of FIG. 3, from left to right, the key genetic elements are as follows: hybrid 5′ long terminal repeat (RSV/LTR), Psi sequence (RNA packaging site), RRE (Rev-response element), cPPT (polypurine tract), a prothrombin enhancer, a hAAT promoter, a PAH sequence including, in embodiments, a codon-optimized PAH sequence or variant thereof, as detailed herein, Woodchuck Post-Transcriptional Regulatory Element (WPRE), and LTR with a deletion in the U3 region.

Referring next to Vector B of FIG. 3, from left to right, the key genetic elements are as follows: hybrid 5′ long terminal repeat (RSV/LTR), Psi sequence (RNA packaging site), RRE (Rev-response element), cPPT (polypurine tract), one HNF1/HNF4 (hepatocyte nuclear factor) binding site upstream of a prothrombin enhancer, a hAAT promoter, a PAH sequence including, in embodiments, a codon-optimized PAH sequence or variant thereof, as detailed herein, a Woodchuck Post-Transcriptional Regulatory Element (WPRE), and LTR with a deletion in the U3 region.

Referring next to Vector C of FIG. 3, from left to right, the key genetic elements are as follows: hybrid 5′ long terminal repeat (RSV/LTR), Psi sequence (RNA packaging site), RRE (Rev-response element), cPPT (polypurine tract), three HNF1/4 (hepatocyte nuclear factor) binding sites upstream of a prothrombin enhancer, a hAAT promoter, a PAH sequence including, in embodiments, a codon-optimized PAH sequence or variant thereof, as detailed herein, a Woodchuck Post-Transcriptional Regulatory Element (WPRE), and LTR with a deletion in the U3 region.

Referring next to Vector D of FIG. 3, from left to right, the key genetic elements are as follows: hybrid 5′ long terminal repeat (RSV/LTR), Psi sequence (RNA packaging site), RRE (Rev-response element), cPPT (polypurine tract), five HNF1 (hepatocyte nuclear factor) binding sites upstream of a prothrombin enhancer, a hAAT promoter, a PAH sequence including, in embodiments, a codon-optimized PAH sequence or variant thereof, as detailed herein, a Woodchuck Post-Transcriptional Regulatory Element (WPRE), and LTR with a deletion in the U3 region.

To produce the vectors outlined generally in FIG. 3, the methods and materials described herein and as otherwise as understood by those skilled in the art were employed.

Inhibitory RNA Design: The sequence of Homo sapiens phenylalanine hydroxylase (PAH) (NM_000277.1) mRNA was used to search for potential shRNA candidates to knockdown PAH levels in human cells. Potential RNA shRNA sequences were chosen from candidates selected by siRNA or shRNA design programs such as from the GPP Web Portal hosted by the Broad Institute (portals.broadinstitute.org/gpp/public/) or the BLOCK-iT RNAi Designer from Thermo Scientific (https://maidesigner.thermofisher.com/maiexpress/). Individual selected shRNA sequences were inserted into a lentiviral vector immediately 3 prime to a RNA polymerase III promoter H1 (H1 Promoter) (SEQ ID NO: 20) to regulate shRNA expression. These lentivirus shRNA constructs were used to transduce cells and measure the change in specific mRNA levels.

Vector Construction: To synthesize shRNA sequences that targeted PAH, oligonucleotide sequences containing BamHI and EcoRI restriction sites were synthesized by Eurofins MWG Operon. Overlapping sense and antisense oligonucleotide sequences were mixed and annealed during cooling from 70 degrees Celsius to room temperature. The lentiviral vector was digested with the restriction enzymes BamHI and EcoRI for one hour at 37 degrees Celsius. The digested lentiviral vector was purified by agarose gel electrophoresis and extracted from the gel using a DNA gel extraction kit from Thermo Scientific. The DNA concentrations were determined and vector to oligo (3:1 ratio) were mixed, allowed to anneal, and ligated. The ligation reaction was performed with T4 DNA ligase for 30 minutes at room temperature. 2.5 microliters of the ligation mix were added to 25 microliters of STBL3 competent bacterial cells. Transformation was achieved after heat-shock at 42 degrees Celsius. Bacterial cells were spread on agar plates containing ampicillin and drug-resistant colonies (indicating the presence of ampicillin-resistance plasmids) were recovered and expanded in LB broth. To check for insertion of the oligo sequences, plasmid DNA was extracted from harvested bacteria cultures with the Thermo Scientific DNA mini prep kit. Insertion of shRNA sequences in the lentiviral vector was verified by DNA sequencing using a specific primer for the promoter used to regulate shRNA expression. Using the following coding sequences, exemplary shRNA sequences were determined to knock-down PAH.

PAH shRNA sequence #1:

(SEQ ID NO: 11)

TCGCATTTCATCAAGATTAATCTCGAG

ATTAATCTTGATGAAATGCGATTTTT

PAH shRNA sequence #2:

(SEQ ID NO: 12)

ACTCATAAAGGAGCATATAAGCTCGAG

CTTATATGCTCCTTTATGAGTTTTTT

Example 3. Liver Specific Prothrombin Enhancer/hAAT Promoter

Hepa1-6 mouse hepatoma and Hep3B human carcinoma cells were transduced with lentiviral vectors containing a liver-specific prothrombin enhancer (SEQ ID NO: 3), and a human alpha-1 anti-trypsin promoter (SEQ ID NO: 4) to create a DNA fragment containing a prothrombin enhancer and a human alpha-1 anti-trypsin promoter. The resulting DNA sequence is as follows: GCGAGAACTTGTGCCTCCCCGTGTCCTGCTCTTTGTCCCTCTGTCCTACTAGAC TAATATTTTGCCTGGGTACTGCAAACAGGAAATGGGGGAGGGACAGGAGTAGGG CGGAGGGTAGCCCGGGGATCTGCTACCAGTGGAACAGCCACTAAGGATTCTGC AGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCAC GCCACCCCCTCCACCTTGGACACAGGACGCTGTGGCTGAGCCAGGTACAATG ACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGG GCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTAGCCCCTGTTTGCTC CTCCGATAACTGGGGTGACCTTGGTAATATCACCAGCAGCCTCCCCCGTTGCC CCTCTGGATCCACTGCTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCT CAGGCACCACCACTGACCTGGGACAGTGAAT (SEQ ID NO: 61). Results for these infections are detailed in further Examples herein.

Example 4. hAAT Promoter with Prothrombin Enhancer and Hepatocyte Nuclear Factor (HNF) Binding Sites

Hepa1-6 mouse hepatoma and Hep3B human carcinoma cells were transduced with lentiviral vectors containing a liver-specific prothrombin enhancer (SEQ ID NO: 3), a human alpha-1 anti-trypsin promoter (SEQ ID NO: 4), and one or more hepatocyte nuclear factor (HNF) binding sites. The resulting DNA sequence that includes a DNA fragment containing a prothrombin enhancer, a human alpha-1 anti-trypsin promoter, and five HNF1 binding sites (designated in underlined font) was as follows:

(SEQ ID NO: 62)

GTTAATCATTAACGTTAATCATTAACGTTAATCAT

TAACGTTAATCATTAACGTTAATCATTAACATCGA

TGCGAGAACTTGTGCCTCCCCGTGTTCCTGCTCTT

TGTCCCTCTGTCCTACTTAGACTAATATTTGCCTT

GGGTACTGCAAACAGGAAATGGGGGAGGGACAGGA

GTAGGGCGGAGGGTAGGATTCTGCAGTGAGAGCAG

AGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTC

TGACTCACGCCACCCCCTCCACCTTGGACACAGGA

CGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTT

TCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGG

CAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGA

TCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTC

CGATAACTGGGGTGACCTTGGTTAATATTCACCAG

CAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTT

AAATACGGACGAGGACAGGGCCCTGTCTCCTCAGC

TTCAGGCACCACCACTGACCTGGGACAGTGAAT.

The resulting DNA sequence that includes a DNA fragment containing a prothrombin enhancer, a human alpha-1 anti-trypsin promoter, and one HNF1/HNF4 binding site (HNF1 designated in underlined font; HNF4 designated in bold font) is as follows:

(SEQ ID NO: 77)

GTTAATCATTAAC
GCTTGTACTTTGGTACAATCGA

TGCGAGAACTTGTGCCTCCCCGTGTTCCTGCTCTT

TGTCCCTCTGTCCTACTTAGACTAATATTTGCCTT

GGGTACTGCAAACAGGAAATGGGGGAGGGACAGGA

GTAGGGCGGAGGGTAGCCCGGGGATTCTGCAGTGA

GAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAG

ACTGTCTGACTCACGCCACCCCCTCCACCTTGGAC

ACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGA

CTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTG

CCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGA

CTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTG

CTCCTCCGATAACTGGGGTGACCTTGGTTAATATT

CACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCA

CTGCTTAAATACGGACGAGGACAGGGCCCTGTCTC

CTCAGCTTCAGGCACCACCACTGACCTGGGACAGT

GAAT.

The resulting DNA sequence that includes a DNA fragment containing a prothrombin enhancer, a human alpha-1 anti-trypsin promoter, and three HNF1/HNF4 binding sites (HNF1 designated in underlined font; HNF4 designated in bold font) is as follows:

(SEQ ID NO: 63)

GTTAATCATTAAC
GCTTGTACTTTGGTACA
GTTAA

TCATTAAC
GCTTGTACTTTGGTACA
GTTAATCATT

AAC
GCTTGTACTTTGGTACAATCGATGCGAGAACT

TGTGCCTCCCCGTGTTCCTGCTCTTTGTCCCTCTG

TCCTACTTAGACTAATATTTGCCTTGGGTACTGCA

AACAGGAAATGGGGGAGGGACAGGAGTAGGGCGGA

GGGTAGCCCGGGGATTCTGCAGTGAGAGCAGAGGG

CCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGAC

TCACGCCACCCCCTCCACCTTGGACACAGGACGCT

GTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGG

TAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAA

GCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC

AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGAT

AACTGGGGTGACCTTGGTTAATATTCACCAGCAGC

CTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAAT

ACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCA

GGCACCACCACTGACCTGGGACAGTGAAT.

The expression of codon-optimized PAH from these vectors is detailed in further Examples herein.

Example 5. Materials and Methods for Synthesizing Vectors Containing PAH

The sequence of Homo sapiens phenylalanine hydroxylase (hPAH) miRNA (Gen Bank: NM_000277.1) was chemically synthesized with EcoRI and Sail restriction enzyme sites located at distal and proximal ends of the gene by Eurofins Genomics (Louisville, Ky.). hPAH treated with EcoRI and SalI restriction enzymes was ligated into the pCDH lentiviral plasmids (System Biosciences, CA) under control of a hybrid promoter comprising parts of ApoE (NM_000001.11, U35114.1) or prothrombin (AF478696.1), and hAAT (HG98385.1) locus control regions.

The lentiviral vector and hPAH sequences were digested with the restriction enzymes BamHI and EcoRI (NEB, Ipswich, Mass.) for two hours at 37 degrees Celsius. The digested lentiviral vector was purified by agarose gel electrophoresis and extracted from the gel using a DNA gel extraction kit from ThermoFisher (Waltham, Mass.). The DNA concentration was determined and then mixed with the PAH sequence using an insert to vector ratio of 3:1. The mixture was ligated with T4 DNA ligase (NEB) for 30 minutes at room temperature. 2.5 microliters of the ligation mix were added to 25 microliters of STBL3 competent bacterial cells (ThermoFisher). Transformation was carried out by heat-shock at 42 degrees Celsius. Bacterial cells were streaked onto agar plates containing ampicillin and then colonies were expanded in LB broth. To check for insertion of the PAH sequences, Plasmid DNA was extracted from harvested bacteria cultures with the ThermoFisher DNA mini prep kit. Insertion of the PAH sequence in the lentiviral vector (LV) was verified by DNA sequencing (Eurofins Genomics). Next, the ApoE enhancer/hAAT promoter or prothrombin enhancer/hAAT promoter sequences with ClaI and EcoRI restriction sites were synthesized by Eurofins Genomics. The lentiviral vector containing a PAH coding sequence and the hybrid promoters were digested with ClaI and EcoRI enzymes and ligated together. The plasmids containing the hybrid promoters were verified by DNA sequencing. The lentiviral vector containing hPAH and a hybrid promoter sequence were then used to package lentiviral particles to test for their ability to express PAH in transduced cells. Mammalian cells were transduced with lentiviral particles. Cells were collected after 3 days and protein was analyzed by immunoblot for PAH expression.

Regulation of the hPAH Sequence:

A liver specific enhancer-promoter was added to the lentiviral vector to regulate PAH expression in a liver-specific manner. Specifically, the prothrombin enhancer was combined with the human alpha-1-anti-trypsin promoter in the lentiviral vector to regulate PAH expression. Restricting transgene expression to liver cells is an important consideration for vector safety and target specificity for a genetic medicine to treat phenylketonuria.

Example 6. Synthesis of Codon-Optimized PAH Sequences

Certain bases within codons were changed in the Homo sapiens phenylalanine hydroxylase (hPAH) mRNA (Gen Bank: NM_000277.1) sequence to create the OPT2 PAH sequence (SEQ ID NO: 2) and OPT3 PAH codon-optimized sequence (SEQ ID NO: 70). The OPT2 and OPT3 PAH sequences flanked with EcoRI and SalI restriction sites were synthesized by Eurofins Genomics and IDT and ligated into a lentiviral vector digested with EcoRI and SalI.

Hybrid PAH codon-optimized sequences were constructed by restriction endonuclease digestion with StuI (New England Biolabs). A C-terminal fragment was digested from the LV-Pro-hAAT-PAH plasmid containing either the OPT2 or OPT3 sequences. The C-terminal OPT3 fragment was ligated back to the plasmid containing the N-terminal OPT2 sequence to create the OPT2/3 sequence (SEQ ID NO: 71). The C-terminal OPT2 sequence was ligated back to the plasmid containing the N-terminal OPT3 sequence to create the OPT3/2 sequence (SEQ ID NO: 72). The correct orientation of the fragments was verified by sequencing (Eurofins Genomics).

Example 7. Expression of PAH with LV-Pro-hAAT-hPAH Expressing Codon-Optimized Versions of PAH in Hepa1-6 Cells

This Example illustrates the expression of PAH using lentiviral vectors that contain Pro hAAT and codon-optimized versions of PAH.

As described in Example 6, hPAH was codon-optimized (GeneArt Thermo and IDT), synthesized (IDT and Eurofins Genomics), and inserted into a lentiviral vector containing the prothrombin enhancer-hAAT promoter. Insertion of the sequences was verified by DNA sequencing (Eurofins Genomics).

Lentiviral vectors containing hPAH or a codon-optimized hPAH were then used to transduce mouse Hepa1-6 cells (American Type Culture Collection). Cells were transduced with lentiviral particles at a multiplicity of infection (MOI) of 5 and after 3 days protein expression was analyzed by immunoblot for PAH expression. Cells were lysed with a Tris-HCl (pH 7.5) buffer containing 1% NP-40 and protease inhibitor mix (Thermo). The cell lysate was centrifuged at 10000 RPM for 15 minutes and protein concentration was determined with the Protein Assay Reagent (Bio-Rad). Protein lysate was separated on a 4-12% Tris-Bis gel (Thermo) and transferred for 12 hours in a Bio-Rad transfer unit. The expression of PAH was detected by immunoblot using an anti-PAH antibody (MilliporeSigma) and an anti-beta actin antibody (MilliporeSigma) was used for the loading control. PAH expression was driven by a prothrombin enhancer and a hAAT promoter. The lentiviral vectors incorporated, in various instances, either a hPAH or codon-optimized version of the hPAH gene.

FIG. 4A depicts data demonstrating PAH expression from a lentiviral vector containing prothrombin-hAAT PAH and prothrombin-hAAT codon-optimized PAH (OPT2; SEQ ID NO: 2) in Hepa1-6 cells. The expression of the codon-optimized version of PAH (OPT2) was 44% less than the expression of hPAH. FIG. 4B compares PAH protein expression by immunoblot from a lentiviral vector containing either prothrombin-hAAT PAH or three different codon-optimized versions of PAH in Hepa1-6 cells. The first lane of the immunoblot consists of un-transduced cells, the second lane is cells transduced with a lentivirus expressing the human version of PAH (hPAH) (set at 1), the third lane is cells transduced with a lentivirus expressing codon-optimized version 3 (OPT3; SEQ ID NO: 70) of PAH (2.6 fold increase), the fourth lane is cells transduced with a lentivirus expressing codon-optimized version 2/3 (OPT2/3; SEQ ID NO: 71) of PAH (1.9 fold increase), and the last lane is cells transduced with a lentivirus expressing codon-optimized version 3/2 (OPT3/2; SEQ ID NO: 72) of PAH (1.4 fold increase). The band intensity for each immunoblot was determined by densitometry using Adobe PhotoShop.

As shown in FIGS. 4A and 4B, transduction with the codon-optimized OPT3 PAH sequence resulted in increased PAH expression (i) relative to transduction with the codon-optimized OPT2 (SEQ ID NO: 2), OPT2/3 (SEQ ID NO: 71), and OPT3/2 PAH (SEQ ID NO: 72) sequences and (ii) relative to transduction with the hPAH sequence (SEQ ID NO: 1).

Example 8. Measuring Expression Levels of PAH mRNA after Transduction of hPAH and Codon-Optimized Versions of PAH in Hepa1-6 Cells

This Example illustrates that expression of PAH RNA is increased in Hepa1-6 carcinoma cells transduced at a MOI of 5 with a lentiviral vector containing prothrombin-hAAT codon-optimized PAH (OPT3 (SEQ ID NO: 70) and OPT2/3 (SEQ ID NO: 71)) relative to a PAH sequence that has not been codon-optimized (SEQ ID NO: 1), as shown in FIG. 5.

hPAH was codon-optimized (GeneArt Thermo), synthesized (IDT and Eurofins Genomics), and inserted into a lentiviral vector containing the prothrombin enhancer-hAAT promoter. Insertion of the sequences was verified by DNA sequencing (Eurofins Genomics). Lentiviral vectors containing non-optimized PAH or codon-optimized PAH were used to transduce Hepa1-6 mouse carcinoma cells (American Type Culture Collection). Cells were transduced with lentiviral particles and after 3 days RNA was extracted with the RNeasy kit (Qiagen) and analyzed by qPCR with a QuantStudio 3 (Thermo). hPAH RNA expression was detected with TaqMan probes and primers (IDT): hPAH FAM TaqMan probe (5′-TCGTGAAAGCTCATGGACAGTGGC-3′: SEQ ID NO: 64) and primer set (PAH TaqMan Forward Primer: 5′-AGATCTTGAGGCATGACATTGG-3′: SEQ ID NO: 65; and PAH TaqMan Reverse Primer: 5′-GTCCAGCTCTTGAATGGTTCT-3′: SEQ ID NO: 66) for hPAH. Total RNA (100 ng) was normalized with an actin FAM probe (5′-AGCGGGAAATCGTGCGTGAC-3′: SEQ ID NO: 67) and primer set (Actin Forward Primer: 5′-GGACCTGACTGACTACCTCAT-3′: SEQ ID NO: 68; and Actin Reverse Primers: 5′-CGTAGCACAGCTTCTCCTTAAT-3′: SEQ ID NO: 69).

As shown in FIG. 5, three groups are compared: Hepa1-6 cells transduced with a lentiviral vector expressing the coding region of PAH (SEQ ID NO: 1) (bar 1) or codon-optimized versions of PAH (OPT3 (SEQ ID NO: 70) and OPT2/3 (SEQ ID NO: 71, bars 2 and 3, respectively) at 5 MOI. PAH RNA levels are expressed as RNA fold change from Hepa1-6 cells expressing PAH (SEQ ID NO: 1) (set at 1). In cells expressing PAH from the codon-optimized version (OPT3: SEQ ID NO: 70), there was a 4.5-fold increase in expression as compared with PAH (SEQ ID NO: 1). In cells expressing PAH from the codon-optimized version (OPT2/3: SEQ ID NO: 71), there was a 2.2-fold increase in expression as compared with PAH (SEQ ID NO: 1).

Example 9. Lentivirus-Delivered Expression of PAH with a Codon-Optimized PAH Sequence and the Prothrombin Enhancer Containing HNF1 or HNF1/4 Binding Sites in Hepa1-6 and Hep3B Cells

This Example illustrates that expression of codon-optimized hPAH is increased in mouse Hepa1-6 and human Hep3B carcinoma cells when transduced with a lentiviral vector containing the hAAT promoter in combination with the prothrombin enhancer and upstream HNF1/4 binding sites, as shown in FIGS. 6A-6B. This example also shows that a codon-optimized version of the hPAH coding sequence (OPT3) expresses more than the non-optimized hPAH coding region sequence in Hepa1-6 cells and Hep3B cells. This Example further illustrates that a lentiviral vector expressing Hepatocyte Nuclear Factor-1 and -4 (HNF1 and HNF1/4) binding sites in combination with the prothrombin enhancer increases the levels of PAH protein in Hepa1-6 cells and Hep3B cells.

hPAH (optimized and non-optimized) and variations of the prothrombin enhancer with HNF1/4 binding sites were synthesized (Eurofin Genomics and IDT) and inserted into a lentiviral vector containing the hAAT promoter. Insertion of the sequences was verified by DNA sequencing (Eurofin Genomics). The lentiviral vectors containing a verified PAH sequence were then used to transduce Hepa1-6 mouse liver cancer cells (American Type Culture Collection, Manassas). Cells were transduced with lentiviral particles at a MOI of 5 and after 3 days protein were analyzed by immunoblot for PAH expression. Cells were lysed with a Tris-HCl (pH 7.5) buffer containing 1% NP-40 and protease inhibitor mix (Thermo). The cell lysate was centrifuged at 10000 RPM for 15 minutes and protein concentration was determined with the Protein Assay Reagent (Bio-Rad). Protein lysate was separated on a 4-12% Tris-Bis gel (Thermo) and transferred for 12 hours in a Bio-Rad transfer unit. The expression of PAH was detected by immunoblot using an anti-PAH antibody (MilliporeSigma) and an anti-beta actin antibody (MilliporeSigma) was used for the loading control. PAH expression was driven by a prothrombin enhancer and a hAAT promoter. The lentiviral vectors incorporated, in various instances, either codon-optimized versions of the hPAH gene or hPAH genes in which the codons remained unaltered. In addition, PAH expression in these constructs was driven by the hAAT promoter containing the liver-specific prothrombin enhancer with upstream HNF1 or HNF1/4 binding sites. The band intensity for the immunoblots were determined by densitometry using Adobe PhotoShop.

As shown in FIG. 6A, six groups are compared: (1) Hepa1-6 cells alone (lane 1), (2) a lentiviral vector expressing the coding region of hPAH by the prothrombin enhancer/hAAT promoter (lane 2) (Set at 1), (3) a lentiviral vector expressing codon-optimized hPAH (OPT3) by the prothrombin enhancer/hAAT promoter (lane 3) (increase of 5.7-fold), (4) a lentiviral vector expressing codon-optimized hPAH (OPT3) by the prothrombin enhancer/hAAT with one HNF-1 and -4 binding site upstream of the prothrombin enhancer (lane 4) (increase of 5.6-fold), (5) a lentiviral vector expressing codon-optimized hPAH (OPT3) by the prothrombin enhancer/hAAT with three HNF-1 and -4 binding sites upstream of the prothrombin enhancer (lane 5) (increase of 5.8-fold), and (6) a lentiviral vector expressing codon-optimized hPAH (OPT3) by the prothrombin enhancer/hAAT with five HNF-1 binding sites upstream of the prothrombin enhancer (lane 6) (increase of 5.9-fold). The sequence for the hPAH used in this experiment was SEQ ID NO: 1. The sequence used for the codon-optimized PAH used in this experiment was SEQ ID NO: 70.

As shown in FIG. 6B, six groups are compared: (1) Hep3B cells alone (lane 1), (2) a lentiviral vector expressing the coding region of hPAH (SEQ ID NO: 1) by the prothrombin enhancer/hAAT promoter (SEQ ID NO: 61) (lane 2) (set at 1), (3) a lentiviral vector expressing codon-optimized hPAH (OPT3) (SEQ ID NO: 70) by the prothrombin enhancer/hAAT promoter (SEQ ID NO: 61) (lane 3) (increase of 4.1-fold), (4) a lentiviral vector expressing codon-optimized hPAH (OPT3) by the prothrombin enhancer/hAAT promoter with one HNF-1 and -4 binding site (SEQ ID NO: 9) upstream of the prothrombin enhancer (lane 4) (increase of 5.3-fold), (5) a lentiviral vector expressing codon-optimized hPAH (OPT3) by the prothrombin enhancer/hAAT promoter with three HNF-1 and -4 binding sites (SEQ ID NO: 10) upstream of the prothrombin enhancer (lane 5) (increase of 4.8-fold), and (6) a lentiviral vector expressing codon-optimized hPAH (OPT3) by the prothrombin enhancer/hAAT promoter with five HNF-1 binding sites (SEQ ID NO: 8) upstream of the prothrombin enhancer (lane 6) (increase of 4.5-fold).

FIGS. 6A and 6B demonstrate that expression of PAH is increased in Hepa1-6 and Hep3B carcinoma cells when transduced by lentiviral vectors containing a codon-optimized version of PAH (OPT3) that have HNF1 or HNF1/4 binding sites upstream of the prothrombin enhancer versus Hepa1-6 and Hep3B carcinoma cells transduced with PAH.

Example 10. Lentivirus-Delivered Expression of hPAH in Huh-7 Cells with a Codon-Optimized PAH Sequence and a Regulatory Sequence Containing Either a hAAT Enhancer/Transthyretin Promoter/Minute Virus of Mouse Intron or a Prothrombin Enhancer/hAAT Promoter/Minute Virus of Mouse Intron

This Example illustrates that expression of codon-optimized human PAH is increased in human hepatocellular carcinoma cells with a lentiviral vector containing liver-specific regulatory elements in comparison to alternative constructs containing introns and alternative enhancer/promoter combinations, as shown in FIG. 7.

The hAAT promoter in combination with the prothrombin enhancer (SEQ ID NO: 61) increased PAH expression, but the addition of an intron sequence from the Minute Virus of Mouse (SEQ ID NO: 80) did not enhance expression. The combination of a prothrombin enhancer and hAAT promoter (SEQ ID NO: 61) with a codon-optimized PAH sequence (SEQ ID NO: 70) resulted in higher expression of PAH as compared with a hAAT promoter (SEQ ID NO: 82) and transthyretin enhancer (SEQ ID NO: 81).

The liver-specific regulatory sequences were synthesized (IDT) and inserted into a lentiviral vector upstream of the optimized PAH sequence. Insertion of the sequences was verified by DNA sequencing (Eurofin Genomics). The lentiviral vectors containing verified sequences were then used to transduce Huh-7 hepatocellular cancer cells (Japanese Collection of Research Bioresources Cell Bank). Cells were transduced with lentiviral particles at a MOI of 50 and after 3 days protein was analyzed by immunoblot for PAH expression. Cells were lysed with a Tris-HCl (pH 7.5) buffer containing 1% NP-40 and protease inhibitor mix (Thermo). The cell lysate was centrifuged at 12,000 RPM for 15 minutes and the protein concentration was determined with the Protein Assay Reagent (Bio-Rad). Protein lysate was separated on a 4-12% Tris-Bis gel (Thermo) and transferred for 16 hours in a Bio-Rad transfer unit. The expression of PAH was detected by immunoblot using an anti-PAH antibody (MilliporeSigma) and an anti-beta actin antibody (MilliporeSigma) was used for the loading control. The band intensity for the immunoblots was determined by densitometry using Adobe PhotoShop.

As shown in FIG. 7, four groups are compared: (i) Huh-7 cells alone (lane 1); (ii) a lentiviral vector expressing codon-optimized hPAH (OPT3; SEQ ID NO: 70) and the prothrombin enhancer/hAAT promoter (SEQ ID NO: 61) (lane 2) (baseline band intensity set at 1); (iii) a lentiviral vector expressing codon-optimized hPAH (OPT3) by a prothrombin enhancer/hAAT promoter and intron sequence of the Minute Virus of Mouse (SEQ ID NO: 78) (lane 3) (band intensity of 0.80); and (iv) a lentiviral vector expressing codon-optimized hPAH (OPT3) by a hAAT promoter/transthyretin enhancer and intron sequence of the Minute Virus of Mouse (SEQ ID NO: 79) (lane 4) (band intensity of 0.36).

The results illustrate that lentiviral vectors encoding an intron sequence from the Minute Virus of Mouse resulted in lower PAH expression relative to lentiviral vectors that lacked this intron sequence (compare lane 2 with lane 3, of FIG. 7). This finding is unexpected because previous research suggests that the intron sequence from the Minute Virus of Mouse increases exogenous gene expression from vectors. In addition, this example unexpectedly shows that lentiviral vectors containing promoter/enhancer combinations used for liver-specific gene expression, resulted in lower PAH expression than lentiviral vectors containing the specific combination of Prothrombin enhancer/hAAT promoter with no additional intron as provided herein (compare lane 2 with lane 4, of FIG. 7).

Example 11. Lentivirus-Delivered Expression of hPAH in Huh-7 Cells with a Codon-Optimized PAH Sequence with Either a Mutant WPRE Sequence or Short WPRE (WPREs) Sequence and Containing Either a PAH or Albumin 3′ UTR Sequence

This Example illustrates that expression of codon-optimized human PAH is increased in human hepatocellular carcinoma cells with a lentiviral vector containing liver-specific regulatory elements in comparison to alternative vector constructs comprising 3′UTRs and alternative WPRE sequences, as shown in FIG. 8.

When the WPRE was modified to a shorter, mutant version without the X-protein sequence (SEQ ID NO: 87), the expression of PAH was less than but similar to the vector containing the wild-type WPRE (SEQ ID NO: 18). When a 3′ UTR sequence from either the PAH gene (SEQ ID NO: 85) or albumin gene (SEQ ID NO: 86) was added downstream of the PAH coding sequence, which resulted in either the PAH optimized version 3-PAH 3′UTR sequence (SEQ ID NO: 83) or the PAH optimized version 3-Albumin 3′UTR sequence (SEQ ID NO: 84), there was decreased expression of PAH relative to the vector that did not contain a 3′UTR.

The WPREs and 3′ UTR sequences were synthesized (IDT) and inserted into a lentiviral vector upstream of the optimized PAH sequence. Insertion of the sequences was verified by DNA sequencing (Eurofin Genomics). The lentiviral vectors containing verified sequences were then used to transduce Huh-7 hepatocellular cancer cells (Japanese Collection of Research Bioresources Cell Bank). Cells were transduced with lentiviral particles at a MOI of 50 and after 3 days protein was analyzed by immunoblot for PAH expression. Cells were lysed with a Tris-HCl (pH 7.5) buffer containing 1% NP-40 and protease inhibitor mix (Thermo). The cell lysate was centrifuged at 12,000 RPM for 15 minutes and the protein concentration was determined with the Protein Assay Reagent (Bio-Rad). Protein lysate was separated on a 4-12% Tris-Bis gel (Thermo) and transferred for 16 hours in a Bio-Rad transfer unit. The expression of PAH was detected by immunoblot using an anti-PAH antibody (MilliporeSigma) and an anti-beta actin antibody (MilliporeSigma) was used for the loading control. The band intensity for the immunoblots was determined by densitometry using Adobe PhotoShop.

As shown in FIG. 8, five groups are compared: (i) Huh-7 cells alone (lane 1); (ii) a lentiviral vector expressing codon-optimized hPAH (OPT3; SEQ ID NO: 70), a prothrombin enhancer/hAAT promoter (SEQ ID NO: 61), and a wild-type WPRE (SEQ ID NO: 18) (lane 2) (baseline band intensity set at 1); (iii) a lentiviral vector expressing codon-optimized hPAH (OPT3; SEQ ID NO: 70), a prothrombin enhancer/hAAT promoter (SEQ ID NO: 61), and a mutant WPRE lacking expression of the X-protein (SEQ ID NO: 87) (lane 3) (band intensity of 0.81); (iv) a lentiviral vector expressing codon-optimized hPAH (OPT3; SEQ ID NO: 70), a prothrombin enhancer/hAAT promoter (SEQ ID NO: 61), and with a PAH 3′ UTR (SEQ ID NO: 85) (lane 4) (band intensity of 0.68); and (v) a lentiviral vector expressing codon-optimized hPAH (OPT3; SEQ ID NO: 70) and a prothrombin enhancer/hAAT promoter (SEQ ID NO: 61) and with a albumin 3′ UTR (SEQ ID NO: 86) (lane 5) (band intensity of 0.85).

The results illustrate that lentiviral vectors substituting a mutant WPRE for the normally used wild-type WPRE, or adding the natural 3′ UTR of human PAH gene, or adding a 3′ UTR from the human albumin gene, that are then used for cell transduction, results in lower expression of PAH compared to the Pro-hAAT-PAH(OPT3) vector containing wild-type WPRE and no 3′ UTR sequence. The results also illustrate the negative effect on PAH expression using a lentiviral vector that encodes natural human PAH 3′UTR relative to a lentiviral vector that encodes an albumin PAH 3′UTR (compare lane 4 with lane 5, of FIG. 8). This finding may be due to a change in secondary structure of the PAH mRNA that results when using the albumin PAH 3′UTR versus the natural human PAH 3′UTR. This change in secondary structure may be reducing the interactions between the coding region of PAH and the 3′UTR, thereby resulting in higher PAH expression levels. Moreover, as shown in this example, when a lentiviral vector is used that lacks a 3′UTR PAH, expression levels of PAH are the highest (compare lanes 4 and 5 with lane 2, of FIG. 8).

Sequence Listing

SEQ

ID

NO:
Description
Sequence

1
hPAH
ATGTCCACTGCGGTC

CTGGAAAACCCAGGC

TTGGGCAGGAAACTC

TCTGACTTTGGACAG

GAAACAAGCTATATT

GAAGACAACTGCAAT

CAAAATGGTGCCATA

TCACTGATCTTCTCA

CTCAAAGAAGAAGTT

GGTGCATTGGCCAAA

GTATTGCGCTTATTT

GAGGAGAATGATGTA

AACCTGACCCACATT

GAATCTAGACCTTCT

CGTTTAAAGAAAGAT

GAGTATGAATTTTTC

ACCCATTTGGATAAA

CGTAGCCTGCCTGCT

CTGACAAACATCATC

AAGATCTTGAGGCAT

GACATTGGTGCCACT

GTCCATGAGCTTTCA

CGAGATAAGAAGAAA

GACACAGTGCCCTGG

TTCCCAAGAACCATT

CAAGAGCTGGACAGA

TTTGCCAATCAGATT

CTCAGCTATGGAGCG

GAACTGGATGCTGAC

CACCCTGGTTTTAAA

GATCCTGTGTACCGT

GCAAGACGGAAGCAG

TTTGCTGACATTGCC

TACAACTACCGCCAT

GGGCAGCCCATCCCT

CGAGTGGAATACATG

GAGGAAGAAAAGAAA

ACATGGGGCACAGTG

TTCAAGACTCTGAAG

TCCTTGTATAAAACC

CATGCTTGCTATGAG

TACAATCACATTTTT

CCACTTCTTGAAAAG

TACTGTGGCTTCCAT

GAAGATAACATTCCC

CAGCTGGAAGACGTT

TCTCAATTCCTGCAG

ACTTGCACTGGTTTC

CGCCTCCGACCTGTG

GCTGGCCTGCTTTCC

TCTCGGGATTTCTTG

GGTGGCCTGGCCTTC

CGAGTCTTCCACTGC

ACACAGTACATCAGA

CATGGATCCAAGCCC

ATGTATACCCCCGAA

CCTGACATCTGCCAT

GAGCTGTTGGGACAT

GTGCCCTTGTTTTCA

GATCGCAGCTTTGCC

CAGTTTTCCCAGGAA

ATTGGCCTTGCCTCT

CTGGGTGCACCTGAT

GAATACATTGAAAAG

CTCGCCACAATTTAC

TGGTTTACTGTGGAG

TTTGGGCTCTGCAAA

CAAGGAGACTCCATA

AAGGCATATGGTGCT

GGGCTCCTGTCATCC

TTTGGTGAATTACAG

TACTGCTTATCAGAG

AAGCCAAAGCTTCTC

CCCCTGGAGCTGGAG

AAGACAGCCATCCAA

AATTACACTGTCACG

GAGTTCCAGCCCCTG

TATTACGTGGCAGAG

AGTTTTAATGATGCC

AAGGAGAAAGTAAGG

AACTTTGCTGCCACA

ATACCTCGGCCCTTC

TCAGTTCGCTACGAC

CCATACACCCAAAGG

ATTGAGGTCTTGGAC

AATACCCAGCAGCTT

AAGATTTTGGCTGAT

TCCATTAACAGTGAA

ATTGGAATCCTTTGC

AGTGCCCTCCAGAAA

ATAAAGTAA

2
Codon-
ATGAGTACGGCTGTG

optimized
CTCGAGAATCCAGGT

PAH (Opt2)
TTGGGCCGAAAGCTG

TCTGATTTTGGACAG

GAGACATCTTATATT

GAAGACAACTGCAAC

CAGAATGGTGCGATA

TCCCTTATTTTTTCT

CTGAAAGAAGAAGTA

GGTGCGCTGGCAAAG

GTCTTGCGGCTGTTT

GAAGAGAACGATGTT

AATCTTACTCATATT

GAGTCCAGACCATCA

CGGCTGAAAAAAGAC

GAGTACGAATTTTTT

ACTCACTTGGACAAA

CGAAGCTTGCCGGCT

CTTACTAATATCATT

AAGATCCTCCGGCAT

GACATAGGGGCGACA

GTGCATGAGCTTTCA

AGGGATAAAAAGAAA

GATACCGTCCCCTGG

TTTCCAAGGACCATA

CAAGAACTCGACCGA

TTCGCGAACCAGATC

CTTTCATATGGTGCT

GAGTTGGATGCTGAC

CACCCCGGCTTCAAA

GACCCGGTCTACCGA

GCGCGGCGGAAACAA

TTTGCTGACATCGCA

TACAATTACAGGCAT

GGCCAGCCAATTCCT

AGAGTAGAATACATG

GAAGAAGAGAAAAAA

ACCTGGGGTACCGTC

TTCAAGACGCTGAAA

TCATTGTATAAAACT

CATGCATGTTACGAA

TATAACCATATTTTT

CCGTTGCTCGAGAAA

TATTGCGGGTTCCAC

GAAGATAACATCCCA

CAACTCGAGGATGTA

TCTCAGTTCCTCCAG

ACCTGTACGGGGTTT

CGACTTAGGCCTGTC

GCGGGTTTGCTCAGT

TCTCGAGACTTCCTG

GGTGGATTGGCGTTT

CGGGTATTCCATTGC

ACGCAGTATATCCGA

CACGGAAGTAAGCCA

ATGTACACGCCAGA

ACCCGATATCTGTCA

CGAATTGCTTGGACA

CGTTCCTCTGTTTTC

TGATCGATCATTCGC

TCAGTTTTCACAGGA

AATCGGCCTGGCATC

TTTGGGAGCGCCGGA

TGAATATATTGAGAA

GCTCGCTACAATTTA

CTGGTTCACGGTAGA

ATTTGGGTTGTGCAA

GCAGGGTGATAGTAT

TAAAGCATACGGTGC

GGGATTGCTGTCCTC

ATTCGGGGAGCTTCA

GTATTGCCTGTCCGA

GAAACCCAAGCTGTT

GCCGTTGGAATTGGA

AAAAACCGCTATCCA

AAATTACACAGTAAC

GGAGTTCCAACCTTT

GTACTACGTAGCCGA

GTCATTTAACGATGC

AAAGGAGAAGGTCAG

AAATTTTGCTGCGAC

GATACCCAGACCGTT

CTCAGTAAGGTACGA

TCCTTACACTCAGAG

GATTGAAGTCCTGGA

TAATACGCAACAGCT

CAAGATCCTGGCAGA

CTCCATAAATTCTGA

AATCGGCATCTTGTG

TTCAGCACTGCAAAA

GATAAAATAA

3
Prothrombin
GCGAGAACTTGTGCC

enhancer(Pro)
TCCCCGTGTTCCTGC

TCTTTGTCCCTCTGT

CCTACTTAGACTAAT

ATTTGCCTTGGGTAC

TGCAAACAGGAAATG

GGGGAGGGACAGGAG

TAGGGCGGAGGGTAG

4
Human alpha-
GATCTTGCTACCAGT

1 anti-trypsin
GGAACAGCCACTAAG

promoter
GATTCTGCAGTGAGA

(hAAT)
GCAGAGGGCCAGCTA

AGTGGTACTCTCCCA

GAGACTGTCTGACTC

ACGCCACCCCCTCCA

CCTTGGACACAGGAC

GCTGTGGTTTCTGAG

CCAGGTACAATGACT

CCTTTCGGTAAGTGC

AGTGGAAGCTGTACA

CTGCCCAGGCAAAGC

GTCCGGGCAGCGTAG

GCGGGCGACTCAGAT

CCCAGCCAGTGGACT

TAGCCCCTGTTTGCT

CCTCCGATAACTGGG

GTGACCTTGGTTAAT

ATTCACCAGCAGCCT

CCCCCGTTGCCCCTC

TGGATCCACTGCTTA

AATACGGACGAGGAC

AGGGCCCTGTCTCCT

CAGCTTCAGGCACCA

CCACTGACCTGGGAC

AGTGAAT

5
Rabbit beta
GTGAGTTTGGGGACC

globin intron
CTTGATTGTTCTTTC

TTTTTCGCTATTGTA

AAATTCATGTTATAT

GGAGGGGGCAAAGTT

TTCAGGGTGTTGTTT

AGAATGGGAAGATGT

CCCTTGTATCACCAT

GGACCCTCATGATAA

TTTTGTTTCTTTCAC

TTTCTACTCTGTTGA

CAACCATTGTCTCCT

CTTATTTTCTTTTCA

TTTTCTGTAACTTTT

TCGTTAAACTTTAGC

TTGCATTTGTAACGA

ATTTTTAAATTCACT

TTTGTTTATTTGTCA

GATTGTAAGTACTTT

CTAGCACAGTTTTAG

AGAACAATTGTTATA

ATTAAATGATAAGGT

AGAATATTTCTGCAT

ATAAATTCTGGCTGG

CGTGGAAATATTCTT

ATTGGTAGAAACAAC

TACACCCTGGTCATC

ATCCTGCCTTTCTCT

TTATGGTTACAATGA

TATACACTGTTTGAG

ATGAGGATAAAATAC

TCTGAGTCCAAACCG

GGCCCCTCTGCTAAC

CATGTTCATGCCTTC

TTCTCTTTCCTACAG

6
Human beta
GGATCCTGAGAACTT

globin intron
CAGGGTGAGTCTATG

GGACGCTTGATGTTT

TCTTTCCCCTTCTTT

TCTATGGTTAAGTTC

ATGTCATAGGAAGGG

GATAAGTAACAGGGT

ACACATATTGACCAA

ATCAGGGTAATTTTG

CATTTGTAATTTTAA

AAAATGCTTTCTTCT

TTTAATATACTTTTT

TGTTTATCTTATTTC

TAATACTTTCCCTAA

TCTCTTTCTTTCAGG

GCAATAATGATACAA

TGTATCATGCCTCTT

TGCACCATTCTAAAG

AATAACAGTGATAAT

TTCTGGGTTAAGGCA

ATAGCAATATTTCTG

CATATAAATATTTCT

GCATATAAATTGTAA

CTGATGTAAGAGGTT

TCATATTGCTAATAG

CAGCTACAATCCAGC

TACCATTCTGCTTTT

ATTTTATGGTTGGGA

TAAGGCTGGATTATT

CTGAGTCCAAGCTAG

GCCCTTTTGCTAATC

ATGTTCATACCTCTT

ATCTTCCTCCCACAG

CTCCTGGGCAACGTG

CTGGTCTGTGTGCTG

GCCCATCACTTTGGC

AAAG

7
IX
GTTAATCATTAAC

Hepatocyte

Nuclear Factor

1 (1XHNFI)

8
5XHcpatocyte
GTTAATCATTAACGT

Nuclear Factor
TAATCATTAACGTTA

1 (5XHNFI)
ATCATTAACGTTAAT

CATTAACGTTAATCA

TTAAC

9
IXHepatocvtc
GTTAATCATTAACGC

Nuclear Factor
TTGTACTTTGGTACA

1/4(IXHNF1/4)

10
3XHepatocvtc
GTTAATCATTAACGC

Nuclear Factor
TTGTACTTTGGTACA

1/4(3XHNF1/4)
GTTAATCATTAACGC

TTGTACTTTGGTACA

GTTAATCATTAACGC

TTGTACTTTGGTACA

11
PAH shRNA
TCGCATTTCATCAAG

sequence #1
ATTAATCTCGAGATT

AATCTTGATGAAATG

CGATTTTT

12
PAH shRNA
ACTCATAAAGGAGCA

sequence #2
TATAAGCTCGAGCTT

ATATGCTCCTTTATG

AGTTTTTT

13
Rous Sarcoma
GTAGTCTTATGCAAT

virus (RSV)
ACTCTTGTAGTCTTG

promoter
CAACATGGTAACGAT

GAGTTAGCAACATGC

CTTACAAGGAGAGAA

AAAGCACCGTGCATG

CCGATTGGTGGAAGT

AAGGTGGTACGATCG

TGCCTTATTAGGAAG

GCAACAGACGGGTCT

GACATGGATTGGACG

AACCACTGAATTGCC

GCATTGCAGAGATAT

TGTATTTAAGTGCCT

AGCTCGATACAATAA

ACG

14
5′ Long
GGTCTCTCTGGTTAG

terminal
ACCAGATCTGAGCCT

repeal (LTR)
GGGAGCTCTCTGGCT

AACTAGGGAACCCAC

TGCTTAAGCCTCAAT

AAAGCTTGCCTTGAG

TGCTTCAAGTAGTGT

GTGCCCGTCTGTTGT

GTGACTCTGGTAACT

AGAGATCCCTCAGAC

CCTTTTAGTCAGTGT

GGAAAATCTCTAGCA

15
Psi Packaging
TACGCCAAAAATTTT

signal (RNA
GACTAGCGGAGGCTA

packaging
GAAGGAGAGAG

site)

16
Rev response
AGGAGCTTTGTTCCT

element(RRE)
TGGGTTCTTGGGAGC

AGCAGGAAGCACTAT

GGGCGCAGCCTCAAT

GACGCTGACGGTACA

GGCCAGACAATTATT

GTCTGGTATAGTGCA

GCAGCAGAACAATTT

GCTGAGGGCTATTGA

GGCGCAACAGCATCT

GTTGCAACTCACAGT

CTGGGGCATCAAGCA

GCTCCAGGCAAGAAT

CCTGGCTGTGGAAAG

ATACCTAAAGGATCA

ACAGCTCC

17
Central
TTTTAAAAGAAAAGG

poly purine
GGGGATTGGGGGGTA

tract (cPPT)
CAGTGCAGGGGAAAG

(poly purine
AATAGTAGACATAAT

tract)
AGCAACAGACATACA

AACTAAAGAATTACA

AAAACAAATTACAAA

ATTCAAAATTTTA

18
Long WPRE
AATCAACCTCTGGAT

sequence
TACAAAATTTGTGAA

AGATTGACTGGTATT

CTTAACTATGTTGCT

CCTTTTACGCTATGT

GGATACGCTGCTTTA

ATGCCTTTGTATCAT

GCTATTGCTTCCCGT

ATGGCTTTCATTTTC

TCCTCCTTGTATAAA

TCCTGGTTGCTGTCT

CTTTATGAGGAGTTG

TGGCCCGTTGTCAGG

CAACGTGGCGTGGTG

TGCACTGTGTTTGCT

GACGCAACCCCCACT

GGTTGGGGCATTGCC

ACCACCTGTCAGCTC

CTTTCCGGGACTTTC

GCTTTCCCCCTCCCT

ATTGCCACGGCGGAA

CTCATCGCCGCCTGC

CTTGCCCGCTGCTGG

ACAGGGGCTCGGCTG

TTGGGCACTGACAAT

TCCGTGGTGTTGTCG

GGGAAATCATCGTCC

TTTCCTTGGCTGCTC

GCCTGTGTTGCCACC

TGGATTCTGCGCGGG

ACGTCCTTCTGCTAC

GTCCCTTCGGCCCTC

AATCCAGCGGACCTT

CCTTCCCGCGGCCTG

CTGCCGGCTCTGC

GGCCTCTTCCGCGTC

TTCGCCTTCGCCCTC

AGACGAGTCGGATCT

CCCTTTGGGCCGCCT

CCCCGCCTG

19
delta U3
TGGAAGGGCTAATTC

3′LTR
ACTCCCAACGAAGAT

AAGATCTGCTTTTTG

CTTGTACTGGGTCTC

TCTGGTTAGACCAGA

TCTGAGCCTGGGAGC

TCTCTGGCTAACTAG

GGAACCCACTGCTTA

AGCCTCAATAAAGCT

TGCCTTGAGTGCTTC

AAGTAGTGTGTGCCC

GTCTGTTGTGTGACT

CTGGTAACTAGAGAT

CCCTCAGACCCTTTT

AGTCAGTGTGGAAAA

TCTCTAGCAGTAGTA

GTTCATGTCA

20
H1 Promoter
GAACGCTGACGTCAT

CAACCCGCTCCAAGG

AATCGCGGGCCCAGT

GTCACTAGGCGGGAA

CACCCAGCGCGCGTG

CGCCCTGGCAGGAAG

ATGGCTGTGAGGGAC

AGGGGAGTGGCGCCC

TGCAATATTTGCATG

TCGCTATGTGTTCTG

GGAAATCACCATAAA

CGTGAAATGTCTTTG

GATTTGGGAATCTTA

TAAGTTCTGTATGAG

ACCACTT

21
CMV
TAGTTATTAATAGTA

enhancer/
ATCAATTACGGGGTC

chicken beta
ATTAGTTCATAGCCC

actin
ATATATGGAGTTCCG

promoter
CGTTACATAACTTAC

GGTAAATGGCCCGCC

TGGCTGACCGCCCAA

CGACCCCCGCCCATT

GACGTCAATAATGAC

GTATGTTCCCATAGT

AACGCCAATAGGGAC

TTTCCATTGACGTCA

ATGGGTGGACTATTT

ACGGTAAACTGCCCA

CTTGGCAGTACATCA

AGTGTATCATATGCC

AAGTACGCCCCCTAT

TGACGTCAATGACGG

TAAATGGCCCGCCTG

GCATTATGCCCAGTA

CATGACCTTATGGGA

CTTTCCTACTTGGCA

GTACATCTACGTATT

AGTCATCGCTATTAC

CATGGGTCGAGGTGA

GCCCCACGTTCTGCT

TCACTCTCCCCATCT

CCCCCCCCTCCCCAC

CCCCAATTTTGTATT

TATTTATTTTTTAAT

TATTTTGTGCAGCGA

TGGGGGCGGGGGGGG

GGGGGGCGCGCGCCA

GGCGGGGCGGGGCGG

GGCGAGGGGCGGGGC

GGGGCGAGGCGGAGA

GGTGCGGCGGCAGCC

AATCAGAGCGGCGCG

CTCCGAAAGTTTCCT

TTTATGGCGAGGCGG

CGGCGGCGGCGGCCC

TATAAAAAGCGAAGC

GCGCGGCGGGCG

22
HIV Gag
ATGGGTGCGAGAGCG

TCAGTATTAAGCGGG

GGAGAATTAGATCGA

TGGGAAAAAATTCGG

TTAAGGCCAGGGGGA

AAGAAAAAATATAAA

TTAAAACATATAGTA

TGGGCAAGCAGGGAG

CTAGAACGATTCGCA

GTTAATCCTGGCCTG

TTAGAAACATCAGAA

GGCTGTAGACAAATA

CTGGGACAGCTACAA

CCATCCCTTCAGACA

GGATCAGAAGAACTT

AGATCATTATATAAT

ACAGTAGCAACCCTC

TATTGTGTGCATCAA

AGGATAGAGATAAAA

GACACCAAGGAAGCT

TTAGACAAGATAGAG

GAAGAGCAAAACAAA

AGTAAGAAAAAAGCA

CAGCAAGCAGCAGCT

GACACAGGACACAGC

AATCAGGTCAGCCAA

AATTACCCTATAGTG

CAGAACATCCAGGGG

CAAATGGTACATCAG

GCCATATCACCTAGA

ACTTTAAATGCATGG

GTAAAAGTAGTAGAA

GAGAAGGCTTTCAGC

CCAGAAGTGATACCC

ATGTTTTCAGCATTA

TCAGAAGGAGCCACC

CCACAAGATTTAAAC

ACCATGCTAAACACA

GTGGGGGGACATCAA

GCAGCCATGCAAATG

TTAAAAGAGACCATC

AATGAGGAAGCTGCA

GAATGGGATAGAGTG

CATCCAGTGCATGCA

GGGCCTATTGCACCA

GGCCAGATGAGAGAA

CCAAGGGGAAGTGAC

ATAGCAGGAACTACT

AGTACCCTTCAGGAA

CAAATAGGATGGATG

ACACATAATCCACCT

ATCCCAGTAGGAGAA

ATCTATAAAAGATGG

ATAATCCTGGGATTA

AATAAAATAGTAAGA

ATGTATAGCCCTACC

AGCATTCTGGACATA

AGACAAGGACCAAAG

GAACCCTTTAGAGAC

TATGTAGACCGATTC

TATAAAACTCTAAGA

GCCGAGCAAGCTTCA

CAAGAGGTAAAAAAT

TGGATGACAGAAACC

TTGTTGGTCCAAAAT

GCGAACCCAGATTGT

AAGACTATTTTAAAA

GCATTGGGACCAGGA

GCGACACTAGAAGAA

ATGATGACAGCATGT

CAGGGAGTGGGGGGA

CCCGGCCATAAAGCA

AGAGTTTTGGCTGAA

GCAATGAGCCAAGTA

ACAAATCCAGCTACC

ATAATGATACAGAAA

GGCAATTTTAGGAAC

CAAAGAAAGACTGTT

AAGTGTTTCAATTGT

GGCAAAGAAGGGCAC

ATAGCCAAAAATTGC

AGGGCCCCTAGGAAA

AAGGGCTGTTGGA

AATGTGGAAAGGAAG

GACACCAAATGAAAG

ATTGTACTGAGAGAC

AGGCTAATTTTTTAG

GGAAGATCTGGCCTT

CCCACAAGGGAAGGC

CAGGGAATTTTCTTC

AGAGCAGACCAGAGC

CAACAGCCCCACCAG

AAGAGAGCTTCAGGT

TTGGGGAAGAGACAA

CAACTCCCTCTCAGA

AGCAGGAGCCGATA

GACAAGGAACTGTAT

CCTTTAGCTTCCCTC

AGATCACTCTTTGGC

AGCGACCCCTCGTCA

CAATAA

23
HIV Pol
ATGAATTTGCCAGGA

AGATGGAAACCAAAA

ATGATAGGGGGAATT

GGAGGTTTTATCAAA

GTAGGACAGTATGAT

CAGATACTCATAGAA

ATCTGCGGACATAAA

GCTATAGGTACAGTA

TTAGTAGGACCTACA

CCTGTCAACATAATT

GGAAGAAATCTGTTG

ACTCAGATTGGCTGC

ACTTTAAATTTTCCC

ATTAGTCCTATTGAG

ACTGTACCAGTAAAA

TTAAAGCCAGGAATG

GATGGCCCAAAAGTT

AAACAATGGCCATTG

ACAGAAGAAAAAATA

AAAGCATTAGTAGAA

ATTTGTACAGAAATG

GAAAAGGAAGGAAAA

ATTTCAAAAATTGGG

CCTGAAAATCCATAC

AATACTCCAGTATTT

GCCATAAAGAAAAAA

GACAGTACTAAATGG

AGAAAATTAGTAGAT

TTCAGAGAACTTAAT

AAGAGAACTCAAGAT

TTCTGGGAAGTTCAA

TTAGGAATACCACAT

CCTGCAGGGTTAAAA

CAGAAAAAATCAGTA

ACAGTACTGGATGTG

GGCGATGCATATTTT

TCAGTTCCCTTAGAT

AAAGACTTCAGGAAG

TATACTGCATTTACC

ATACCTAGTATAAAC

AATGAGACACCAGGG

ATTAGATATCAGTAC

AATGTGCTTCCACAG

GGATGGAAAGGATCA

CCAGCAATATTCCAG

TGTAGCATGACAAAA

ATCTTAGAGCCTTTT

AGAAAACAAAATCCA

GACATAGTCATCTAT

CAATACATGGATGAT

TTGTATGTAGGATCT

GACTTAGAAATAGGG

CAGCATAGAACAAAA

ATAGAGGAACTGAGA

CAACATCTGTTGAGG

TGGGGATTTACCACA

CCAGACAAAAAACAT

CAGAAAGAACCTCCA

TTCCTTTGGATGGGT

TATGAACTCCATCCT

GATAAATGGACAGTA

CAGCCTATAGTGCTG

CCAGAAAAGGACAGC

TGGACTGTCAATGAC

ATACAGAAATTAGTG

GGAAAATTGAATTGG

GCAAGTCAGATTTAT

GCAGGGATTAAAGTA

AGGCAATTATGTAAA

CTTCTTAGGGGAACC

AAAGCACTAACAGAA

GTAGTACCACTAACA

GAAGAAGCAGAGCTA

GAACTGGCAGAAAAC

AGGGAGATTCTAAAA

GAACCGGTACATGGA

GTGTATTATGACCCA

TCAAAAGACTTAATA

GCAGAAATACAGAAG

CAGGGGCAAGGCCAA

TGGACATATCAAATT

TATCAAGAGCCATTT

AAAAATCTGAAAACA

GGAAAATATGCAAGA

ATGAAGGGTGCCCAC

ACTAATGATGTGAAA

CAATTAACAGAGGCA

GTACAAAAAATAGCC

ACAGAAAGCATAGTA

ATATGGGGAAAGACT

CCTAAATTTAAATTA

CCCATACAAAAGGAA

ACATGGGAAGCATGG

TGGACAGAGTATTGG

CAAGCCACCTGGATT

CCTGAGTGGGAGTTT

GTCAATACCCCTCCC

TTAGTGAAGTTATGG

TACCAGTTAGAGAAA

GAACCCATAATAGGA

GCAGAAACTTTCTAT

GTAGATGGGGCAGCC

AATAGGGAAACTAAA

TTAGGAAAAGCAGGA

TATGTAACTGACAGA

GGAAGACAAAAAGTT

GTCCCCCTAACGGAC

ACAACAAATCAGAAG

ACTGAGTTACAAGCA

ATTCATCTAGCTTTG

CAGGATTCGGGATTA

GAAGTAAACATAGTG

ACAGACTCACAATAT

GCATTGGGAATCATT

CAAGCACAACCAGAT

AAGAGTGAATCAGAG

TTAGTCAGTCAAATA

ATAGAGCAGTTAATA

AAAAAGGAAAAAGTC

TACCTGGCATGGGTA

CCAGCACACAAAGGA

ATTGGAGGAAATGAA

CAAGTAGATGGGTTG

GTCAGTGCTGGAATC

AGGAAAGTACTA

24
HIV Integrase
TTTTTAGATGGAATA

(HIV Int)
GATAAGGCCCAAGAA

GAACATGAGAAATAT

CACAGTAATTGGAGA

GCAATGGCTAGTGAT

TTTAACCTACCACCT

GTAGTAGCAAAAGAA

ATAGTAGCCAGCTGT

GATAAATGTCAGCTA

AAAGGGGAAGCCATG

CATGGACAAGTAGAC

TGTAGCCCAGGAATA

TGGCAGCTAGATTGT

ACACATTTAGAAGGA

AAAGTTATCTTGGTA

GCAGTTCATGTAGCC

AGTGGATATATAGAA

GCAGAAGTAATTCCA

GCAGAGACAGGGCAA

GAAACAGCATACTTC

CTCTTAAAATTAGCA

GGAAGATGGCCAGTA

AAAACAGTACATACA

GACAATGGCAGCAAT

TTCACCAGTA

CTACAGTTAAGGCCG

CCTGTTGGTGGGCGG

GGATCAAGCAGGAAT

TTGGCATTCCCTACA

ATCCCCAAAGTCAAG

GAGTAATAGAATCTA

TGAATAAAGAATTAA

AGAAAATTATAGGAC

AGGTAAGAGATCAGG

CTGAACATCTTAAGA

CAGCAGTACAAATGG

CAGTATTCATCCACA

ATTTTAAAAGAAAAG

GGGGGATTGGGGGGT

ACAGTGCAGGGGAAA

GAATAGTAGACATAA

TAGCAACAGACATAC

AAACTAAAGAATTAC

AAAAACAAATTACAA

AAATTCAAAATTTTC

GGGTTTATTACAGGG

ACAGCAGAGATCCAG

TTTGGAAAGGACCAG

CAAAGCTCCTCTGGA

AAGGTGAAGGGGCAG

TAGTAATACAAGATA

ATAGTGACATAAAAG

TAGTGCCAAGAAGAA

AAGCAAAGATCATCA

GGGATTATGGAAAAC

AGATGGCAGGTGATG

ATTGTGTGGCAAGTA

GACAGGATGAGGATT

AA

25
HIV RRE
AGGAGCTTTGTTCCT

TGGGTTCTTGGGAGC

AGCAGGAAGCACTAT

GGGCGCAGCGTCAAT

GACGCTGACGGTACA

GGCCAGACAATTATT

GTCTGGTATAGTGCA

GCAGCAGAACAATTT

GCTGAGGGCTATTGA

GGCGCAACAGCATCT

GTTGCAACTCACAGT

CTGGGGCATCAAGCA

GCTCCAGGCAAGAAT

CCTGGCTGTGGAAAG

ATACCTAAAGGATCA

ACAGCTCCT

26
HIV Rev
ATGGCAGGAAGAAGC

GGAGACAGCGACGAA

GAACTCCTCAAGGCA

GTCAGACTCATCAAG

TTTCTCTATCAAAGC

AACCCACCTCCCAAT

CCCGAGGGGACCCGA

CAGGCCCGAAGGAAT

AGAAGAAGAAGGTGG

AGAGAGAGACAGAGA

CAGATCCATTCGATT

AGTGAACGGATCCTT

AGCACTTATCTGGGA

CGATCTGCGGAGCCT

GTGCCTCTTCAGCTA

CCACCGCTTGAGAGA

CTTACTCTTGATTGT

AACGAGGATTGTGGA

ACTTCTGGGACGCAG

GGGGTGGGAAGCCCT

CAAATATTGGTGGAA

TCTCCTACAATATTG

GAGTCAGGAGCTAAA

GAATAG

27
CMV
ACATTGATTATTGAC

Promoter
TAGTTATTAATAGTA

ATCAATTACGGGGTC

ATTAGTTCATAGCCC

ATATATGGAGTTCCG

CGTTACATAACTTAC

GGTAAATGGCCCGCC

TGGCTGACCGCCCAA

CGACCCCCGCCCATT

GACGTCAATAATGAC

GTATGTTCCCATAGT

AACGCCAATAGGGAC

TTTCCATTGACGTCA

ATGGGTGGAGTATTT

ACGGTAAACTGCCCA

CTTGGCAGTACATCA

AGTGTATCATATGCC

AAGTACGCCCCCTAT

TGACGTCAATGACGG

TAAATGGCCCGCCTG

GCATTATGCCCAGTA

CATGACCTTATGGGA

CTTTCCTACTTGGCA

GTACATCTACGTATT

AGTCATCGCTATTAC

CATGGTGATGCGGTT

TTGGCAGTACATCAA

TGGGCGTGGATAGCG

GTTTGACTCACGGGG

ATTTCCAAGTCTCCA

CCCCATTGACGTCAA

TGGGAGTTTGTTTTG

GCACCAAAATCAACG

GGACTTTCCAAAATG

TCGTAACAACTCCGC

CCCATTGACGCAAAT

GGGCGGTAGGCGTG

TACGGTGGGAGGTCT

ATATAAGCAGAGCTC

TCTGGCTAACTAGAG

AACCCACTGCTTACT

G

28
Vesicular
ATGAAGTGCCTTTTG

stomatitis
TACTTAGCCTTTTTA

Indiana virus
TTCATTGGGGTGAAT

glycoprotein
TGCAAGTTCACCATA

VSV-G
GTTTTTCCACACAAC

CAAAAAGGAAACTGG

AAAAATGTTCCTTCT

AATTACCATTATTGC

CCGTCAAGCTCAGAT

TTAAATTGGCATAAT

GACTTAATAGGCACA

GCCTTACAAGTCAAA

ATGCCCAAGAGTCAC

AAGGCTATTCAAGCA

GACGGTTGGATGTGT

CATGCTTCCAAATGG

GTCACTACTTGTGAT

TTCCGCTGGTATGGA

CCGAAGTATATAACA

CATTCCATCCGATCC

TTCACTCCATCTGTA

GAACAATGCAAGGAA

AGCATTGAACAAACG

AAACAAGGAACTTGG

CTGAATCCAGGCTTC

CCTCCTCAAAGTTGT

GGATATGCAACTGTG

ACGGATGCCGAAGCA

GTGATTGTCCAGGTG

ACTCCTCACCATGTG

CTGGTTGATGAATAC

ACAGGAGAATGGGTT

GATTCACAGTTCATC

AACGGAAAATGCAGC

AATTACATATGCCCC

ACTGTCCATAACTCT

ACAACCTGGCATTCT

GACTATAAGGTCAAA

GGGCTATGTGATTCT

AACCTCATTTCCATG

GACATCACCTTCTTC

TCAGAGGACGGAGAG

CTATCATCCCTGGGA

AAGGAGGGCACAGGG

TTCAGAAGTAACTAC

TTTGCTTATGAAACT

GGAGGCAAGGC

CTGCAAAATGCAATA

CTGCAAGCATTGGGG

AGTCAGACTCCCATC

AGGTGTCTGGTTCGA

GATGGCTGATAAGGA

TCTCTTTGCTGCAGC

CAGATTCCCTGAATG

CCCAGAAGGGTCAAG

TATCTCTGCTCCATC

TCAGACCTCAGTGGA

TGTAAGTCTAATTCA

GGACGTTGAGAGGAT

CTTGGATTATTCCCT

CTGCCAAGAAACCTG

GAGCAAAATCAGAGC

GGGTCTTCCAATCTC

TCCAGTGGATCTCAG

CTATCTTGCTCCTAA

AAACCCAGGAACCGG

TCCTGCTTTCACCAT

AATCAATGGTACCCT

AAAATACTTTGAGAC

CAGATACATCAGAGT

CGATATTGCTGCTCC

AATCCTCTCAAGAAT

GGTCGGAATGATCAG

TGGAACTACCACAGA

AAGGGAACTGTGGGA

TGACTGGGCACCATA

TGAAGACGTGGAAAT

TGGACCCAATGGAGT

TCTGAGGACCAGTTC

AGGATATAAGTTTCC

TTTATACATGATTGG

ACATGGTATGTTGGA

CTCCGATCTTCATCT

TAGCTCAAAGGCTCA

GGTGTTCGAACATCC

TCACATTCAAGACGC

TGCTTCGCAACTTCC

TGATGATGAGAGTTT

ATTTTTTGGTGATAC

TGGGCTATCCAAAAA

TCCAATCGAGCTTGT

AGAAGGTTGGTTCAG

TAGTTGGAAAAGCTC

TATTGCCTCTTTTTT

CTTTATCATAGGGTT

AATCATTGGACTATT

CTTGGTTCTCCGAGT

TGGTATCCATCTTTG

CATTAAATTAAAGCA

CACCAAGAAAAGACA

GATTTATACAGACAT

AGAGATGAACCGACT

TGGAAAGTGA

29
Left ITR
CCTGCAGGCAGCTGC

GCGCTCGCTCGCTCA

CTGAGGCCGCCCGGG

CAAAGCCCGGGCGTC

GGGCGACCTTTGGTC

GCCCGGCCTCAGTGA

GCGAGCGAGCGCGCA

GAGAGGGAGTGGCCA

ACTCCATCACTAGGG

GTTCCT

30
Poly A
GACTGTGCCTTCTAG

Element
TTGCCAGCCATCTGT

TGTTTGCCCCTCCCC

CGTGCCTTCCTTGAC

CCTGGAAGGTGCCAC

TCCCACTGTCCTTTC

CTAATAAAATGAGGA

AATTGCATCGCATTG

TCTGAGTAGGTGTCA

TTCTATTCTGGGGGG

TGGGGTGGGGCAGGA

CAGCAAGGGGGAGGA

TTGGGAAGACAATAG

CAGGCATGCTGGGGA

TGCGGTGGGCTCTAT

GGC

31
Right ITR
AGGAACCCCTAGTGA

TGGAGTTGGCCACTC

CCTCTCTGCGCGCTC

GCTCGCTCACTGAGG

CCGGGCGACCAAAGG

TCGCCCGACGCCCGG

GCTTTGCCCGGGCGG

CCTCAGTGAGCGAGC

GAGCGCGCAGCTGCC

TGCAGG

32
E2A Element
TTAAAAGTCGAAGGG

GTTCTCGCGCTCGTC

GTTGTGCGCCGCGCT

GGGGAGGGCCACGTT

GCGGAACTGGTACTT

GGGCTGCCACTTGAA

CTCGGGGATCACCAG

TTTGGGCACTGGGGT

CTCGGGGAAGGTCTC

GCTCCACATGCGCCG

GCTCATCTGCAGGGC

GCCCAGCATGTCAGG

CGCGGAGATCTTGAA

ATCGCAGTTGGGGCC

GGTGCTCTGCGCGCG

CGAGTTGCGGTACAC

TGGGTTGCAGCACTG

GAACACCATCAGACT

GGGGTACTTCACACT

AGCCAGCACGCTCTT

GTCGCTGATCTGATC

CTTGTCCAGGTCCTC

GGCGTTGCTCAGGCC

GAACGGGGTCATCTT

GCACAGCTGGCGGCC

CAGGAAGGGCACGCT

CTGAGGCTTGTGGTT

ACACTCGCAGTGCAC

GGGCATCAGCATCAT

CCCCGCGCCGCGCTG

CATATTCGGGTAGAG

GGCCTTGACGAAGGC

CGCGATCTGCTTGAA

AGCTTGCTGGGCCTT

GGCCCCCTCGCTGAA

AAACAGGCCGCAGCT

CTTCCCGCTGAACTG

ATTATTCCCGCACCC

GGCATCATGGACGCA

GCAGCGCGCGTCATG

GCTGGTCAGTTGCAC

CACGCTCCGTCCCCA

GCGGTTCTGGGTCAC

CTTGGCCTTGCTGGG

TTGCTCCTTCAGCGC

ACGCTGCCCGTTCTC

ACTGGTCACATCCAT

CTCCACCACGTGGTC

CTTGTGGATCATCAC

CGTCCCATGCAGACA

CTTGAGCTGGCCTTC

CACCTCGGTGCAGCC

GTGGTCCCACAGGGC

ACTGCCGGTGCACTC

CCAGTTCTTGTGCGC

GATCCCGCTGTGGCT

GAAGATGTAACCTTG

CAACAGGCGACCCAT

GATGGTGCTAAAGCT

CTTCTGGGTGGTGAA

GGTCAGTTGCAGACC

GCGGGCCTCCTCGTT

CATCCAGGTCTGGCA

CATCTTTTGGAAGAT

CTCGGTCTGCTCGGG

CATGAGCTTGTAAGC

ATCGCGCAGGCCGCT

GTCGACGCGGTAGCG

TTCCATCAGCACATT

CATGGTATCCATGCC

CTTCTCCCAGGACGA

GACCAGAGG

CAGACTCAGGGGGTT

GCGCACGTTCAGGAC

ACCGGGGGTCGCGGG

CTCGACGATGCGTTT

TCCGTCCTTGCCTTC

CTTCAACAGAACCGG

CGGCTGGCTGAATCC

CACTCCCACGATCAC

GGCTTCTTCCTGGGG

CATCTCTTCGTCTGG

GTCTACCTTGGTCAC

ATGCTTGGTCTTTCT

GGCTTGCTCCGGATC

CCACCCGCTGATACT

TTCGGCGCTTGGTTG

GCAGAGGAGGTGGCG

GCGAGGGGCTCCTCT

CCTGCTCCGGCGGAT

AGCGCGCTGAACCGT

GGCCCCGGGGCGGAG

TGGCCTCTCGGTCCA

TGAACCGGCGCACGT

CCTGACTGCCGCCGG

CCAT

33
E4 element
TCATGTATCTTTATT

GATTTTTACACCAGC

ACGGGTAGTCAGTCT

CCCACCACCAGCCCA

TTTCACAGTGTAAAC

AATTCTCTCAGCACG

GGTGGCCTTAAATAG

GGCAATATTCTGATT

AGTGCGGGAACTGGA

CTTGGGGTCTATAAT

CCACACAGTTTCCTG

GCGAGCCAAACGGGG

GTCGGTGATTGAGAT

GAAGCCGTCCTCTGA

AAAGTCATCCAAGCG

AGCCTCACAGTCCAA

GGTCACAGTATTATG

ATAATCTGCATGATC

ACAATCGGGCAACAG

GGGATGTTGTTCAGT

CAGTGAAGCCCTGGT

TTCCTCATCAGATCG

TGGTAAACGGGCCCT

GCGATATGGATGATG

GCGGAGCGAGCTGGA

TTGAATCTCGGTTTG

CAT

34
VARNA
AGCGGGCACTCTTCC

GTGGTCTGGTGGATA

AATTCGCAAGGGTAT

CATGGCGGACGACCG

GGGTTCGAGCCCCGT

ATCCGGCCGTCCGCC

GTGATCCATGCGGTT

ACCGCCCGCGTGTCG

AACCCAGGTGTGCGA

CGTCAGACAACGGGG

GAGTGCTCCTTT

35
AAV2 Rep
ATGGCTGCCGATGGT

TATCTTCCAGATTGG

CTCGAGGACACTCTC

TCTGAAGGAATAAGA

CAGTGGTGGAAGCTC

AAACCTGGCCCACCA

CCACCAAAGCCCGCA

GAGCGGCATAAGGAC

GACAGCAGGGGTCTT

GTGCTTCCTGGGTAC

AAGTACCTCGGACCC

TTCAACGGACTCGAC

AAGGGAGAGCCGGTC

AACGAGGCAGACGCC

GCGGCCCTCGAGCAC

GACAAAGCCTACGAC

CGGCAGCTCGACAGC

GGAGACAACCCGTAC

CTCAAGTACAACCAC

GCCGACGCGGAGTTT

CAGGAGCGCCTTAAA

GAAGATACGTCTTTT

GGGGGCAACCTCGGA

CGAGCAGTCTTCCAG

GCGAAAAAGAGGGTT

CTTGAACCTCTGGGC

CTGGTTGAGGAACCT

GTTAAGACGGCTCCG

GGAAAAAAGAGGCCG

GTAGAGCACTCTCCT

GTGGAGCCAGACTCC

TCCTCGGGAACCGGA

AAGGCGGGCCAGCAG

CCTGCAAGAAAAAGA

TTGAATTTTGGTCAG

ACTGGAGACGCAGAC

TCAGTACCTGACCCC

CAGCCTCTCGGACAG

CCACCAGCAGCCCCC

TCTGGTCTGGGAACT

AATACGATGGCTACA

GGCAGTGGCGCACCA

ATGGCAGACAATAAC

GAGGGCGCCGACGGA

GTGGGTAATTCCTCG

GGAAATTGGCATTGC

GATTCCACATGGATG

GGCGACAGAGTCATC

ACCACCAGCACCCGA

ACCTGGGCCCTGCCC

ACCTACAACAACCAC

CTCTACAAACAAATT

TCCAGCCAATCAGGA

GCCTCGAACGACAAT

CACTACTTTGGCTAC

AGCACCCCTTGGGGG

TATTTTGACTTCAAC

AGATTCCACTGCCAC

TTTTCACCACGTGAC

TGGCAAAGACTCATC

AACAACAACTGGGGA

TTCCGACCCAAGAGA

CTCAACTTCAAGCTC

TTTAACATTCAAGTC

AAAGAGGTCACGCAG

AATGACGGTACGACG

ACGATTGCCAATAAC

CTTACCAGCACGGTT

CAGGTGTTTACTGAC

TCGGAGTACCAGCTC

CCGTACGTCCTCGGC

TCGGCGCATCAAGGA

TGCCTCCCGCCGTTC

CCAGCAGACGTCTTC

ATGGTGCCACAGTAT

GGATACCTCACCCTG

AACAACGGGAGTCAG

GCAGTAGGACGCTCT

TCATTTTACTGCCTG

GAGTACTTTCCTTCT

CAGATGCTGCGTACC

GGAAACAACTTTACC

TTCAGCTACACTTTT

GAGGACGTTCCTTTC

CACAGCAGCTACGCT

CACAGCCAGAGTCTG

GACCGTCTCATGAAT

CCTCTCATCGACCAG

TACCTGTATTACTTG

AGCAGAACAAACACT

CCAAGTGGAACCACC

ACGCAGTCAAGGCTT

CAGTTTTCTCAGGCC

GGAGCGAGTGACATT

CGGGACCAGTCTAGG

AACTGGCTTCCTGGA

CCCTGTTACCGCCAG

CAGCGAGTATCAAAG

ACATCTGCGGATAAC

AAC

AACAGTGAATACTCG

TGGACTGGAGCTACC

AAGTACCACCTCAAT

GGCAGAGACTCTCTG

GTGAATCCGGGCCCG

GCCATGGCAAGCCAC

AAGGAAGCAAGGCTC

AGAGAAAACAAATGT

GGACATTGAAAAGGT

CATGATTACAGACGA

AGAGGAAATCAGGAC

AACCAATCCCGTGGC

TACGGAGCAGTATGG

TTCTGTATCTACCAA

CCTCCAGAGAGGCAA

CAGACAAGCAGCTAC

CGCAGATGTCAACAC

ACAAGGCGTTCTTCC

AGGCATGGTCTGGCA

GGACAGAGATGTGTA

CCTTCAGGGGCCCAT

CTGGGCAAAGATTCC

ACACACGGACGGACA

TTTTCACCCCTCTCC

CCTCATGGGTGGATT

CGGACTTAAACACCC

TCCTCCACAGATTCT

CATCAAGAACACCCC

GGTACCTGCGAATCC

TTCGACCACCTTCAG

TGCGGCAAAGTTTGC

TTCCTTCATCACACA

GTACTCCACGGGACA

GGTCAGCGTGGAGAT

CGAGTGGGAGCTGCA

GAAGGAAAACAGCAA

ACGCTGGAATCCCGA

AATTCAGTACACTTC

CAACTACAACAAGTC

TGTTAATGTGGACTT

TACTGTGGACACTAA

TGGCGTGTATTCAGA

GCCTCGCCCCATTGG

CACCAGATACCTGAC

TCGTAATCTGTAA

36
AAV2 Cap
ATGCCGGGGTTTTAC

GAGATTGTGATTAAG

GTCCCCAGCGACCTT

GACGAGCATCTGCCC

GGCATTTCTGACAGC

TTTGTGAACTGGGTG

GCCGAGAAGGAATGG

GAGTTGCCGCCAGAT

TCTGACATGGATCTG

AATCTGATTGAGCAG

GCACCCCTGACCGTG

GCCGAGAAGCTGCAG

CGCGACTTTCTGACG

GAATGGCGCCGTGTG

AGTAAGGCCCCGGAG

GCCCTTTTCTTTGTG

CAATTTGAGAAGGGA

GAGAGCTACTTCCAC

ATGCACGTGCTCGTG

GAAACCACCGGGGTG

AAATCCATGGTTTTG

GGACGTTTCCTGAGT

CAGATTCGCGAAAAA

CTGATTCAGAGAATT

TACCGCGGGATCGAG

CCGACTTTGCCAAAC

TGGTTCGCGGTCACA

AAGACCAGAAATGGC

GCCGGAGGCGGGAAC

AAGGTGGTGGATGAG

TGCTACATCCCCAAT

TACTTGCTCCCCAAA

ACCCAGCCTGAGCTC

CAGTGGGCGTGGACT

AATATGGAACAGTAT

TTAAGCGCCTGTTTG

AATCTCACGGAGCGT

AAACGGTTGGTGGCG

CAGCATCTGACGCAC

GTGTCGCAGACGCAG

GAGCAGAACAAAGAG

AATCAGAATCCCAAT

TCTGATGCGCCGGTG

ATCAGATCAAAAACT

TCAGCCAGGTACATG

GAGCTGGTCGGGTGG

CTCGTGGACAAGGGG

ATTACCTCGGAGAAG

CAGTGGATCCAGGAG

GACCAGGCCTCATAC

ATCTCCTTCAATGCG

GCCTCCAACTCGCGG

TCCCAAATCAAGGCT

GCCTTGGACAATGCG

GGAAAGATTATGAGC

CTGACTAAAACCGCC

CCCGACTACCTGGTG

GGCCAGCAGCCCGTG

GAGGACATTTCCAGC

AATCGGATTTATAAA

ATTTTGGAACTAAAC

GGGTACGATCCCCAA

TATGCGGCTTCCGTC

TTTCTGGGATGGGCC

ACGAAAAAGTTCGGC

AAGAGGAACACCATC

TGGCTGTTTGGGCCT

GCAACTACCGGGAAG

ACCAACATCGCGGAG

GCCATAGCCCACACT

GTGCCCTTCTACGGG

TGCGTAAACTGGACC

AATGAGAACTTTCCC

TTCAACGACTGTGTC

GACAAGATGGTGATC

TGGTGGGAGGAGGGG

AAGATGACCGCCAAG

GTCGTGGAGTCGGCC

AAAGCCATTCTCGGA

GGAAGCAAGGTGCGC

GTGGACCAGAAATGC

AAGTCCTCGGCCCAG

ATAGACCCGACTCCC

GTGATCGTCACCTCC

AACACCAACATGTGC

GCCGTGATTGACGGG

AACTCAACGACCTTC

GAACACCAGCAGCCG

TTGCAAGACCGGATG

TTCAAATTTGAACTC

ACCCGCCGTCTGGAT

CATGACTTTGGGAAG

GTCACCAAGCAGGAA

GTCAAAGACTTTTTC

CGGTGGGCAAAGGAT

CACGTGGTTGAGGTG

GAGCATGAATTCTAC

GTCAAAAAGGGTGGA

GCCAAGAAAAGACCC

GCCCCCAGTGACGCA

GATATAAGTGAGCCC

AAACGGGTGCGCGAG

TCAGTTGCGCAGCCA

TCGACGTCAGACGCG

GAAGCTTCGATCAAC

TACGCAGACAGGTAC

CAAAACAAATGTTCT

CGTCACGTGGGCATG

AATCTGATGCTGTTT

CCCTGCAGACAATGC

GAGAGAATGAATCAG

AATTCAAATATCTGC

TTCACTCACGGACAG

AAAGACTGTTTAGAG

TGCTTTCCCGTGTCA

GAATCTCAACCCGTT

TCTGTCGTCAAAAAG

GCGTATCAGAAACTG

TGCTACATTCATCAT

ATCATGGGAAAGGTG

CCAGACGCTTG

CACTGCCTGCGATCT

GGTCAATGTGGATTT

GGATGACTGCATCTT

TGAACAATAA

37
AAV8 Cap
ATGGCTGCAGGCGGT

GGCGCACCAATGGCA

GACAATAACGAAGGC

GCCGACGGAGTGGGT

AGTTCCTCGGGAAAT

TGGCATTGCGATTCC

ACATGGCTGGGCGAC

AGAGTCATCACCACC

AGCACCCGAACCTGG

GCCCTGCCCACCTAC

AACAACCACCTCTAC

AAGCAAATCTCCAAC

GGGACATCGGGAGGA

GCCACCAACGACAAC

ACCTACTTCGGCTAC

AGCACCCCCTGGGGG

TATTTTGACTTTAAC

AGATTCCACTGCCAC

TTTTCACCACGTGAC

TGGCAGCGACTCATC

AACAACAACTGGGGA

TTCCGGCCCAAGAGA

CTCAGCTTCAAGCTC

TTCAACATCCAGGTC

AAGGAGGTCACGCAG

AATGAAGGCACCAAG

ACCATCGCCAATAAC

CTCACCAGCACCATC

CAGGTGTTTACGGAC

TCGGAGTACCAGCTG

CCGTACGTTCTCGGC

TCTGCCCACCAGGGC

TGCCTGCCTCCGTTC

CCGGCGGACGTGTTC

ATGATTCCCCAGTAC

GGCTACCTAACACTC

AACAACGGTAGTCAG

GCCGTGGGACGCTCC

TCCTTCTACTGCCTG

GAATACTTTCCTTCG

CAGATGCTGAGAACC

GGCAACAACTTCCAG

TTTACTTACACCTTC

GAGGACGTGCCTTTC

CACAGCAGCTACGCC

CACAGCCAGAGCTTG

GACCGGCTGATGAAT

CCTCTGATTGACCAG

TACCTGTACTACTTG

TCTCGGACTCAAACA

ACAGGAGGCACGGCA

AATACGCAGACTCTG

GGCTTCAGCCAAGGT

GGGCCTAATACAATG

GCCAATCAGGCAAAG

AACTGGCTGCCAGGA

CCCTGTTACCGCCAA

CAACGCGTCTCAACG

ACAACCGGGCAAAAC

AACAATAGCAACTTT

GCCTGGACTGCTGGG

ACCAAATACCATCTG

AATGGAAGAAATTCA

TTGGCTAATCCTGGC

ATCGCTATGGCAACA

CACAAAGACGACGAG

GAGCGTTTTTTTCCC

AGTAACGGGATCCTG

ATTTTTGGCAAACAA

AATGCTGCCAGAGAC

AATGCGGATTACAGC

GATGTCATGCTCACC

AGCGAGGAAGAAATC

AAAACCACTAACCCT

GTGGCTACAGAGGAA

TACGGTATCGTGGCA

GATAACTTGCAGCAG

CAAAACACGGCTCCT

CAAATTGGAACTGTC

AACAGCCAGGGGGCC

TTACCCGGTATGGTC

TGGCAGAACCGGGAC

GTGTACCTGCAGGGT

CCCATCTGGGCCAAG

ATTCCTCACACGGAC

GGCAACTTCCACCCG

TCTCCGCTGATGGGC

GGCTTTGGCCTGAAA

CATCCTCCGCCTCAG

ATCCTGATCAAGAAC

ACGCCTGTACCTGCG

GATCCTCCGACCACC

TTCAACCAGTCAAAG

CTGAACTCTTTCATC

ACGCAATACAGCACC

GGACAGGTCAGCGTG

GAAATTGAATGGGAG

CTGCAGAAGGAAAAC

AGCAAGCGCTGGAAC

CCCGAGATCCAGTAC

ACCTCCAACTACTAC

AAATCTACAAGTGTG

GACTTTGCTGTTAAT

ACAGAAGGCGTGTAC

TCTGAACCCCGCCCC

ATTGGCACCCGTTAC

CTCACCCGTAATCTG

TAA

38
AAV DJ Cap
ATGGCTGCCGATGGT

TATCTTCCAGATTGG

CTCGAGGACACTCTC

TCTGAAGGAATAAGA

CAGTGGTGGAAGCTC

AAACCTGGCCCACCA

CCACCAAAGCCCGCA

GAGCGGCATAAGGAC

GACAGCAGGGGTCTT

GTGCTTCCTGGGTAC

AAGTACCTCGGACCC

TTCAACGGACTCGAC

AAGGGAGAGCCGGTC

AACGAGGCAGACGCC

GCGGCCCTCGAGCAC

GACAAAGCCTACGAC

CGGCAGCTCGACAGC

GGAGACAACCCGTAC

CTCAAGTACAACCAC

GCCGACGCCGAGTT

CCAGGAGCGGCTCAA

AGAAGATACGTCTTT

TGGGGGCAACCTCGG

GCGAGCAGTCTTCCA

GGCCAAAAAGAGGCT

TCTTGAACCTCTTGG

TCTGGTTGAGGAAGC

GGCTAAGACGGCTCC

TGGAAAGAAGAGGCC

TGTAGAGCACTCTCC

TGTGGAGCCAGACTC

CTCCTCGGGAACCGG

AAAGGCGGGCCAGCA

GCCTGCAAGAAAAAG

ATTGAATTTTGGTCA

GACTGGAGACGCAGA

CTCAGTCCCAGACCC

TCAACCAATCGGAGA

ACCTCCCGCAGCCCC

CTCAGGTGTGGGATC

TCTTACAATGGCTGC

AGGCGGTGGCGCACC

AATGGCAGACAATAA

CGAGGGCGCCGACGG

AGTGGGTAATTCCTC

GGGAAATTGGCATTG

CGATTCCACATGGAT

GGGCGACAGAGTCAT

CACCACCAGCACCCG

AACCTG

GGCCCTGCCCACCTA

CAACAACCACCTCTA

CAAGCAAATCTCCAA

CAGCACATCTGGAGG

ATCTTCAAATGACAA

CGCCTACTTCGGCTA

CAGCACCCCCTGGGG

GTATTTTGACTTTAA

CAGATTCCACTGCCA

CTTTTCACCACGTGA

CTGGCAGCGACTCAT

CAACAACAACTGGGG

ATTCCGGCCCAAGAG

ACTCAGCTTCAAGCT

CTTCAACATCCAGGT

CAAGGAGGTCACGCA

GAATGAAGGCACCAA

GACCATCGCCAATAA

CCTCACCAGCACCAT

CCAGGTGTTTACGGA

CTCGGAGTACCAGCT

GCCGTACGTTCTCGG

CTCTGCCCACCAGGG

CTGCCTGCCTCCGTT

CCCGGCGGACGTGTT

CATGATTCCCCAGTA

CGGCTACCTAACACT

CAACAACGGTAGTCA

GGCCGTGGGACGCTC

CTCCTTCTACTGCCT

GGAATACTTTCCTTC

GCAGATGCTGAGAAC

CGGCAACAACTTCCA

GTTTACTTACACCTT

CGAGGACGTGCCTTT

CCACAGCAGCTACGC

CCACAGCCAGAGCTT

GGACCGGCTGATGAA

TCCTCTGATTGACCA

GTACCTGTACTACTT

GTCTCGGACTCAAAC

AACAGGAGGCACGAC

AAATACGCAGACTCT

GGGCTTCAGCCAAGG

TGGGCCTAATACAAT

GGCCAATCAGGCAAA

GAACTGGCTGCCAGG

ACCCTGTTACCGCCA

GCAGCGAGTATCAAA

GACATCTGCGGATAA

CAACAACAGTGAATA

CTCGTGGACTGGAGC

TACCAAGTACCACCT

CAATGGCAGAGACTC

TCTGGTGAATCCGGG

CCCGGCCATGGCAAG

CCACAAGGACGATGA

AGAAAAGTTTTTTCC

TCAGAGCGGGGTTCT

CATCTTTGGGAAGCA

AGGCTCAGAGAAAAC

AAATGTGGACATTGA

AAAGGTCATGATTAC

AGACGAAGAGGAAAT

CAGGACAACCAATCC

CGTGGCTACGGAGCA

GTATGGTTCTGTATC

TACCAACCTCCAGAG

AGGCAACAGACAAGC

AGCTACCGCAGATGT

CAACACACAAGGCGT

TCTTCCAGGCATGGT

CTGGCAGGACAGAGA

TGTGTACCTTCAGGG

GCCCATCTGGGCAAA

GATTCCACACACGGA

CGGACATTTTCACCC

CTCTCCCCTCATGGG

TGGATTCGGACTTAA

ACACCCTCCGCCTCA

GATCCTGATCAAGAA

CACGCCTGTACCTGC

GGATCCTCCGACCAC

CTTCAACCAGTCAAA

GCTGAACTCTTTCAT

CACCCAGTATTCTAC

TGGCCAAGTCAGCGT

GGAGATCGAGTGGGA

GCTGCAGAAGGAAAA

CAGCAAGCGCTGGAA

CCCCGAGATCCAGTA

CACCTCCAACTACTA

CAAATCTACAAGTGT

GGACTTTGCTGTTAA

TACAGAAGGCGTGTA

CTCTGAACCCCGCCC

CATTGGCACCCGTTA

CCTCACCCGTAATCT

GTAA

39
Chicken bela
GGAGTCGCTGCGTTG

actin intron
CCTTCGCCCCGTGCC

CCGCTCCGCGCCGCC

TCGCGCCGCCCGCCC

CGGCTCTGACTGACC

GCGTTACTCCCACAG

GTGAGCGGGCGGGAC

GGCCCTTCTCCTCCG

GGCTGTAATTAGCGC

TTGGTTTAATGACGG

CTCGTTTCTTTTCTG

TGGCTGCGTGAAAGC

CTTAAAGGGCTCCGG

GAGGGCCCTTTGTGC

GGGGGGGAGCGGCTC

GGGGGGTGCGTGCGT

GTGTGTGTGCGTGGG

GAGCGCCGCGTGCGG

CCCGCGCTGCCCGGC

GGCTGTGAGCGCTGC

GGGCGCGGCGCGGGG

CTTTGTGCGCTCCGC

GTGTGCGCGAGGGGA

GCGCGGCCGGGGGCG

GTGCCCCGCGGTGCG

GGGGGGCTGCGAGGG

GAACAAAGGCTGCGT

GCGGGGTGTGTGCGT

GGGGGGGTGAGCAGG

GGGTGTGGGCGCGGC

GGTCGGGCTGTAACC

CCCCCCTGCACCCCC

CTCCCCGAGTTGCTG

AGCACGGCCCGGCTT

CGGGTGCGGGGCTCC

GTGCGGGGCGTGGCG

CGGGGCTCGCCGTGC

CGGGCGGGGGGTGGC

GGCAGGTGGGGGTGC

CGGGCGGGGCGGGGC

CGCCTCGGGCCGGGG

AGGGCTCGGGGGAGG

GGCGCGGCGGCCCCG

GAGCGCCGGCGGCTG

TCGAGGCGCGGCGAG

CCGCAGCCATTGCCT

TTTATGGTAATCGTG

CGAGAGGGCGCAGGG

ACTTCCTTTGTCCCA

AATCTGGCGGAGCCG

AAATCTGGGAGGCGC

CGCCGCACCCCCTCT

AGCGGGCGCGGGCGA

AGCGGTGCGGCGCCG

GCAGGAAGGAAATGG

GCGGGGAGGGCCTT

CGTGCGTCGCCGCGC

CGCCGTCCCCTTCTC

CATCTCCAGCCTCGG

GGCTGCCGCAGGGGG

ACGGCTGCCTTCGGG

GGGGACGGGGCAGGG

CGGGGTTCGGCTTCT

GGCGTGTGACCGGCG

G

40
Rabbit beta
AGATCTTTTTCCCTC

globin pols A
TGCCAAAAATTATGG

GGACATCATGAAGCC

CCTTGAGCATCTGAC

TTCTGGCTAATAAAG

GAAATTTATTTTCAT

TGCAATAGTGTGTTG

GAATTTTTTGTGTCT

CTCACTCGGAAGGAC

ATATGGGAGGGCAAA

TCATTTAAAACATCA

GAATGAGTATTTGGT

TTAGAGTTTGGCAAC

ATATGCCATATGCTG

GCTGCCATGAACAAA

GGTGGCTATAAAGAG

GTCATCAGTATATGA

AACAGCCCCCTGCTG

TCCATTCCTTATTCC

ATAGAAAAGCCTTGA

CTTGAGGTTAGATTT

TTTTTATATTTTGTT

TTGTGTTATTTTTTT

CTTTAACATCCCTAA

AATTTTCCTTACATG

TTTTACTAGCCAGAT

TTTTCCTCCTCTCCT

GACTACTCCCAGTCA

TAGCTGTCCCTCTTC

TCTTATGAAGATC

41
Forward
TAAGCAGAATTCATG

Primer
AATTTGCCAGGAAGA

T

42
Reverse
CCATACAATGAATGG

Primer
ACACTAGGCGGCCGC

ACGAAT

43
Gag, Pol,
GAATTCATGAATTTG

Intcgrasc
CCAGGAAGATGGAAA

fragment
CCAAAAATGATAGGG

GGAATTGGAGGTTTT

ATCAAAGTAAGACAG

TATGATCAGATACTC

ATAGAAATCTGCGGA

CATAAAGCTATAGGT

ACAGTATTAGTAGGA

CCTACACCTGTCAAC

ATAATTGGAAGAAAT

CTGTTGACTCAGATT

GGCTGCACTTTAAAT

TTTCCCATTAGTCCT

ATTGAGACTGTACCA

GTAAAATTAAAGCCA

GGAATGGATGGCCCA

AAAGTTAAACAATGG

CCATTGACAGAAGAA

AAAATAAAAGCATTA

GTAGAAATTTGTACA

GAAATGGAAAAGGAA

GGAAAAATTTCAAAA

ATTGGGCCTGAAAAT

CCATACAATACTCCA

GTATTTGCCATAAAG

AAAAAAGACAGTACT

AAATGGAGAAAATTA

GTAGATTTCAGAGAA

CTTAATAAGAGAACT

CAAGATTTCTGGGAA

GTTCAATTAGGAATA

CCACATCCTGCAGGG

TTAAAACAGAAAAAA

TCAGTAACAGTACTG

GATGTGGGCGATGCA

TATTTTTCAGTTCCC

TTAGATAAAGACTTC

AGGAAGTATACTGCA

TTTACCATACCTAGT

ATAAACAATGAGACA

CCAGGGATTAGATAT

CAGTACAATGTGCTT

CCACAGGGATGGAAA

GGATCACCAGCAATA

TTCCAGTGTAGCATG

ACAAAAATCTTAGAG

CCTTTTAGAAAACAA

AATCCAGACATAGTC

ATCTATCAATACATG

GATGATTTGTATGTA

GGATCTGACTTAGAA

ATAGGGCAGCATAGA

ACAAAAATAGAGGAA

CTGAGACAACATCTG

TTGAGGTGGGGATTT

ACCACACCAGACAAA

AAACATCAGAAAGAA

CCTCCATTCCTTTGG

ATGGGTTATGAACTC

CATCCTGATAAATGG

ACAGTACAGCCTATA

GTGCTGCCAGAAAAG

GACAGCTGGACTGTC

AATGACATACAGAAA

TTAGTGGGAAAATTG

AATTGGGCAAGTCAG

ATTTATGCAGGGATT

AAAGTAAGGCAATTA

TGTAAACTTCTTAGG

GGAACCAAAGCACTA

ACAGAAGTAGTACCA

CTAACAGAAGAAGCA

GAGCTAGAACTGGCA

GAAAACAGGGAGATT

CTAAAAGAACCGGTA

CATGGAGTGTATTAT

GACCCATCAAAAGAC

TTAATAGCAGAAATA

CAGAAGCAGGGGCAA

GGCCAATGGACATAT

CAAATTTATCAAGAG

CCATTTAAAAATCTG

AAAACAGGAAAGTAT

GCAAGAATGAAGGGT

GCCCACACTAATGAT

GTGAAACAATTAACA

GAGGCAGTACAAAAA

ATAGCCACAGAAAGC

ATAGTAATATGGGGA

AAGACTCCTAAATTT

AAATTACCCATACAA

AAGGAAACATGGGAA

GCATGGTGGACAGAG

TATTGGCAAGCCACC

TGGATTCCTGAGTGG

GAGTTTGTCAATACC

CCTCCCTTAGTGAAG

TTATGGTACCAGTTA

GAGAAAGAACCCATA

ATAGGAGCAGAAACT

TTCTATGTAGATGGG

GCAGCCAATAGGGAA

ACTAAATTAGGAAAA

GCAGGATATGTAACT

GACAGAGGAAGACAA

AAAGTTGTCCCCCTA

ACGGACACAACAAAT

CAGAAGACTGAGTTA

CAAGCAATTCATCTA

GCTTTGCAGGATTCG

GGATTAGAAGTAAAC

ATAGTGACAGACTCA

CAATATGCATTGGGA

ATCATTCAAGCACAA

CCAGATAAGAGTGAA

TCAGAGTTAGTCAGT

CAAATAATAGAGCAG

TTAATAAAAAAGGAA

AAAGTCTACCTGGCA

TGGGTACCAGCACAC

AAAGGAATTGGAGGA

AATGAACAAGTAGAT

AAATTGGTCAGTGCT

GGAATCAGGAAAGTA

CTATTTTTAGATGGA

ATAGATAAGGCCCAA

GAAGAACATGAGAAA

TATCACAGTAATTGG

AGAGCA

ATGGCTAGTGATTTT

AACCTACCACCTGTA

GTAGCAAAAGAAATA

GTAGCCAGCTGTGAT

AAATGTCAGCTAAAA

GGGGAAGCCATGCAT

GGACAAGTAGACTGT

AGCCCAGGAATATGG

CAGCTAGATTGTACA

CATTTAGAAGGAAAA

GTTATCTTGGTAGCA

GTTCATGTAGCCAGT

GGATATATAGAAGCA

GAAGTAATTCCAGCA

GAGACAGGGCAAGAA

ACAGCATACTTCCTC

TTAAAATTAGCAGGA

AGATGGCCAGTAAAA

ACAGTACATACAGAC

AATGGCAGCAATTTC

ACCAGTACTACAGTT

AAGGCCGCCTGTTGG

TGGGCGGGGATCAAG

CAGGAATTTGGCATT

CCCTACAATCCCCAA

AGTCAAGGAGTAATA

GAATCTATGAATAAA

GAATTAAAGAAAATT

ATAGGACAGGTAAGA

GATCAGGCTGAACAT

CTTAAGACAGCAGTA

CAAATGGCAGTATTC

ATCCACAATTTTAAA

AGAAAAGGGGGGATT

GGGGGGTACAGTGCA

GGGGAAAGAATAGTA

GACATAATAGCAACA

GACATACAAACTAAA

GAATTACAAAAACAA

ATTACAAAAATTCAA

AATTTTCGGGTTTAT

TACAGGGACAGCAGA

GATCCAGTTTGGAAA

GGACCAGCAAAGCTC

CTCTGGAAAGGTGAA

GGGGCAGTAGTAATA

CAAGATAATAGTGAC

ATAAAAGTAGTGCCA

AGAAGAAAAGCAAAG

ATCATCAGGGATTAT

GGAAAACAGATGGCA

GGTGATGATTGTGTG

GCAAGTAGACAGGAT

GAGGATTAA

44
DNA
TCTAGAATGGCAGGA

Fragment
AGAAGCGGAGACAGC

containing the
GACGAAGAGCTCATC

RRE, REV,
AGAACAGTCAGACTC

and rabbit beta
ATCAAGCTTCTCTAT

globin
CAAAGCAACCCACCT

poly A
CCCAATCCCGAGGGG

sequence
ACCCGACAGGCCCGA

AGGAATAGAAGAAGA

AGGTGGAGAGAGAGA

CAGAGACAGATCCAT

TCGATTAGTGAACGG

ATCCTTGGCACTTAT

CTGGGACGATCTGCG

GAGCCTGTGCCTCTT

CAGCTACCACCGCTT

GAGAGACTTACTCTT

GATTGTAACGAGGAT

TGTGGAACTTCTGGG

ACGCAGGGGGTGGGA

AGCCCTCAAATATTG

GTGGAATCTCCTACA

ATATTGGAGTCAGGA

GCTAAAGAATAGAGG

AGCTTTGTTCCTTGG

GTTCTTGGGAGCAGC

AGGAAGCACTATGGG

CGCAGCGTCAATGAC

GCTGACGGTACAGGC

CAGACAATTATTGTC

TGGTATAGTGCAGCA

GCAGAACAATTTGCT

GAGGGCTATTGAGGC

GCAACAGCATCTGTT

GCAACTCACAGTCTG

GGGCATCAAGCAGCT

CCAGGCAAGAATCCT

GGCTGTGGAAAGATA

CCTAAAGGATCAACA

GCTCCTAGATCTTTT

TCCCTCTGCCAAAAA

TTATGGGGACATCAT

GAAGCCCCTTGAGCA

TCTGACTTCTGGCTA

ATAAAGGAAATTTAT

TTTCATTGCAATAGT

GTGTTGGAATTTTTT

GTGTCTCTCACTCGG

AAGGACATATGGGAG

GGCAAATCATTTAAA

ACATCAGAATGAGTA

TTTGGTTTAGAGTTT

GGCAACATATGCCAT

ATGCTGGCTGCCATG

AACAAAGGTGGCTAT

AAAGAGGTCATCAGT

ATATGAAACAGCCCC

CTGCTGTCCATTCCT

TATTCCATAGAAAAG

CCTTGACTTGAGGTT

AGATTTTTTTTATAT

TTTGTTTTGTGTTAT

TTTTTTCTTTAACAT

CCCTAAAATTTTCCT

TACATGTTTTACTAG

CCAGATTTTTCCTCC

TCTCCTGACTACTCC

CAGTCATAGCTGTCC

CTCTTCTCTTATGAA

GATCCCTCGACCTGC

AGCCCAAGCTTGGCG

TAATCATGGTCATAG

CTGTTTCCTGTGTGA

AATTGTTATCCGCTC

ACAATTCCACACAAC

ATACGAGCCGGAAGC

ATAAAGTGTAAAGCC

TGGGGTGCCTAATGA

GTGAGCTAACTCACA

TTAATTGCGTTGCGC

TCACTGCCCGCTTTC

CAGTCGGGAAACCTG

TCGTGCCAGCGGATC

CGCATCTCAATTAGT

CAGCAACCATAGTCC

CGCCCCTAACTCCGC

CCATCCCGCCCCTAA

CTCCGCCCAGTTCCG

CCCATTCTCCGCCCC

ATGGCTGACTAATTT

TTTTTATTTATGCAG

AGGCCGAGGCCGCCT

CGGCCTCTGAGCTAT

TCCAGAAGTAGTGAG

GAGGCTTTTTTGGAG

GCCTAGGCTTTTGCA

AAAAGCTAACTTGTT

TATTGCAGCTTATAA

TGGTTACAAATAAAG

CAATAGCATCACATC

CAAACTCATCAATGT

ATCTTATCAGCGGCC

GCCCCGGG

45
DNA
ACGCGTTAGTTATTA

fragment
ATAGTAATCAATTAC

Containing
GGGGTCATTAGTTCA

the
TAGCCCATATATGGA

CAG
GTTCCGCGTTACATA

enhancer/
ACTTACGGTAAATGG

promoter
CCCGCCTGGCTGACC

intron
GCCCAACGACCCCCG

sequence
CCCATTGACGTCAAT

AATGACGTATGTTCC

CATAGTAACGCCAAT

AGGGACTTTCCATTG

ACGTCAATGGGTGGA

CTATTTACGGTAAAC

TGCCCACTTGGCAGT

ACATCAAGTGTATCA

TATGCCAAGTACGCC

CCCTATTGACGTCAA

TGACGGTAAATGGCC

CGCCTGGCATTATGC

CCAGTACATGACCTT

ATGGGACTTTCCTAC

TTGGCAGTACATCTA

CGTATTAGTCATCGC

TATTACCATGGGTCG

AGGTGAGCCCCACGT

TCTGCTTCACTCTCC

CCATCTCCCCCCCCT

CCCCACCCCCAATTT

TGTATTTATTTATTT

TTTAATTATTTTGTG

CAGCGATGGGGGCGG

GGGGGGGGGGGGCGC

GCGCCAGGCGGGGCG

GGGCGGGGCGAGGGG

CGGGGCGGGGCGAGG

CGGAGAGGTGCGGCG

GCAGCCAATCAGAGC

GGCGCGCTCCGAAAG

TTTCCTTTTATGGCG

AGGCGGCGGCGGCGG

CGGCCCTATAAAAAG

CGAAGCGCGCGGCGG

GCGGGAGTCGCTGCG

TTGCCTTCGCCCCGT

GCCCCGCTCCGCGCC

GCCTCGCGCCGCCCG

CCCCGGCTCTGACTG

ACCGCGTTACTCCCA

CAGGTGAGCGGGCGG

GACGGCCCTTCTCCT

CCGGGCTGTAATTAG

CGCTTGGTTTAATGA

CGGCTCGTTTCTTTT

CTGTGGCTGCGTGAA

AGCCTTAAAGGGCTC

CGGGAGGGCCCTTTG

TGCGGGGGGGAGCGG

CTCGGGGGGTGCGTG

CGTGTGTGTGTGCGT

GGGGAGCGCCGCGTG

CGGCCCGCGCTGCCC

GGCGGCTGTGAGCGC

TGCGGGCGCGGCGCG

GGGCTTTGTGCGCTC

CGCGTGTGCGCGAGG

GGAGCGCGGCCGGGG

GCGGTGCCCCGCGGT

GCGGGGGGGCTGCGA

GGGGAACAAAGGCTG

CGTGCGGGGTGTGTG

CGTGGGGGGGTGAGC

AGGGGGTGTGGGCGC

GGCGGTCGGGCTGTA

ACCCCCCCCTGCACC

CCCCTCCCCGAGTTG

CTGAGCACGGCCCGG

CTTCGGGTGCGGGGC

TCCGTGCGGGGCGTG

GCGCGGGGCTCGCCG

TGCCGGGCGGGGGGT

GGCGGCAGGTGGGGG

TGCCGGGCGGGGCGG

GGCCGCCTCGGGCCG

GGGAGGGCTCGGGGG

AGGGGCGCGGCGGCC

CCGGAGCGCCGGCGG

CTGTCGAGGCGCGGC

GAGCCGCAGCCATTG

CCTTTTATGGTAATC

GTGCGAGAGGGCGCA

GGGACTTCCTTTGTC

CCAAATCTGGCGGAG

CCGAAATCTGGGAGG

CGCCGCCGCACCCCC

TCTAGCGGGCGCGGG

CGAAGCGGTGCGGCG

CCGGCAGGAAGGAAA

TGGGCGGGGAGGGCC

TTCGTGCGTCGCCGC

GCCGCCGTCCCCTTC

TCCATCTCCAGCCTC

GGGGCTGCCGCAGGG

GGACGGCTGCCTTCG

GGGGGGACGGGGCAG

GGCGGGGTTCGGCTT

CTGGCGTGTGACCGG

CGGGAATTC

46
RSV promoter
CAATTGCGATGTACG

and HIV Rev
GGCCAGATATACGCG

TATCTGAGGGGACTA

GGGTGTGTTTAGGCG

AAAAGCGGGGCTTCG

GTTGTACGCGGTTAG

GAGTCCCCTCAGGAT

ATAGTAGTTTCGCTT

TTGCATAGGGAGGGG

GAAATGTAGTCTTAT

GCAATACACTTGTAG

TCTTGCAACATGGTA

ACGATGAGTTAGCAA

CATGCCTTACAAGGA

GAGAAAAAGCACCGT

GCATGCCGATTGGTG

GAAGTAAGGTGGTAC

GATCGTGCCTTATTA

GGAAGGCAACAGACA

GGTCTGACATGGATT

GGACGAACCACTGAA

TTCCGCATTGCAGAG

ATAATTGTATTTAAG

TGCCTAGCTCGATAC

AATAAACGCCATTTG

ACCATTCACCACATT

GGTGTGCACCTCCAA

GCTCGAGCTCGTTTA

GTGAACCGTCAGATC

GCCTGGAGACGCCAT

CCACGCTGTTTTGAC

CTCCATAGAAGACAC

CGGGACCGATCCAGC

CTCCCCTCGAAGCTA

GCGATTAGGCATCTC

CTATGGCAGGAAGAA

GCGGAGACAGCGACG

AAGAACTCCTCAAGG

CAGTCAGACTCATCA

AGTTTCTCTATCAAA

GCAACCCACCTCCCA

ATCCCGAGGGGACCC

GACAGGCCCGAAGGA

ATAGAAGAAGAAGGT

GGAGAGAGAGACAGA

GACAGATCCATTCGA

TTAGTGAACGGATCC

TTAGCACTTATCTGG

GACGATCTGCGGAGC

CTGTGCCTCTTCAGC

TACCACCGCTTGAGA

GACTTACTCTTGATT

GTAACGAGGATTGTG

GAACTTCTGGGACGC

AGGGGGTGGGAAGCC

CTCAAATATTGGTGG

AATCTCCTACAATAT

TGGAGTCAGGAGCTA

AAGAATAGTCTAGA

47
Elongation
CCGGTGCCTAGAGAA

Factor-1 alpha
GGTGGCGCGGGGTAA

(EF-1 alpha)
ACTGGGAAAGTGATG

promoter
TCGTGTACTGGCTCC

GCCTTTTTCCCGAGG

GTGGGGGAGAACCGT

ATATAAGTGCAGTAG

TCGCCGTGAACGTTC

TTTTTCGCAACGGGT

TTGCCGCCAGAACAC

AGGTAAGTGCCGTGT

GTGGTTCCCGCGGGC

CTGGCCTCTTTACGG

GTTATGGCCCTTGCG

TGCCTTGAATTACTT

CCACGCCCCTGGCTG

CAGTACGTGATTCTT

GATCCCGAGCTTCGG

GTTGGAAGTGGGTGG

GAGAGTTCGAGGCCT

TGCGCTTAAGGAGCC

CCTTCGCCTCGTGCT

TGAGTTGAGGCCTGG

CCTGGGCGCTGGGGC

CGCCGCGTGCGAATC

TGGTGGCACCTTCGC

GCCTGTCTCGCTGCT

TTCGATAAGTCTCTA

GCTAGTCTTGTAAAT

GCGGGGCAAGATCTG

CACACTGGTATTTCG

GTTTTTGGGGCCGCG

GGCGGCGACGGGGCC

CGTGCGTCCCAGCGC

ACATGTTCGGCGAGG

CGGGGCCTGCGAGCG

CGGCCACCGAGAATC

GGACGGGGGTAGTCT

CAAGCTGGCCGGCCT

GCTCTGGTGCCTGGC

CTCGCGCCGCCGTGT

ATCGCCCCGCCCTGG

GCGGCAAGGCTGGCC

CGGTCGGCACCAGTT

GCGTGAGCGGAAAGA

TGGCCGCTTCCCGGC

CCTGCTGCAGGGAGC

TCAAAATGGAGGACG

CGGCGCTCGGGAGAG

CGGGCGGGTGAGTCA

CCCACACAAAGGAAA

AGGGCCTTTCCGTCC

TCAGCCGTCGCTTCA

TGTGACTCCACGGAG

TACCGGGCGCCGTCC

AGGCACCTCGATTAG

TTCTCGAGCTTTTGG

AGTACGTCGTCTTTA

GGTTGGGGGGAGGGG

TTTTATGCGATGGAG

TTTCCCCACACTGAG

TGGGTGGAGACTGAA

GTTAGGCCAGCTTGG

CACTTGATGTAATTC

TCCTTGGAATTTGCC

CTTTTTGAGTTTGGA

TCTTGGTTCATTCTC

AAGCCTCAGACAGTG

GTTCAAAGTTTTTTT

CTTCCATTTCAGGTG

TCGTGA

48
PGK Promoter
GGGGTTGGGGTTGCG

CCTTTTCCAAGGCAG

CCCTGGGTTTGCGCA

GGGACGCGGCTGCTC

TGGGCGTGGTTCCGG

GAAACGCAGCGGCGC

CGACCCTGGGTCTCG

CACATTCTTCACGTC

CGTTCGCAGCGTCAC

CCGGATCTTCGCCGC

TACCCTTGTGGGCCC

CCCGGCGACGCTTCC

TGCTCCGCCCCTAAG

TCGGGAAGGTTCCTT

GCGGTTCGCGGCGTG

CCGGACGTGACAAAC

GGAAGCCGCACGTCT

CACTAGTACCCTCGC

AGACGGACAGCGCCA

GGGAGCAATGGCAGC

GCGCCGACCGCGATG

GGCTGTGGCCAATAG

CGGCTGCTCAGCAGG

GCGCGCCGAGAGCAG

CGGCCGGGAAGGGGC

GGTGCGGGAGGCGGG

GTGTGGGGCGGTAGT

GTGGGCCCTGTTCCT

GCCCGCGCGGTGTTC

CGCATTCTGCAAGCC

TCCGGAGCGCACGTC

GGCAGTCGGCTCCCT

CGTTGACCGAATCAC

CGACCTCTCTCCCCA

G

49
UbC Promoter
GCGCCGGGTTTTGGC

GCCTCCCGCGGGCGC

CCCCCTCCTCACGGC

GAGCGCTGCCACGTC

AGACGAAGGGCGCAG

GAGCGTTCCTGATCC

TTCCGCCCGGACGCT

CAGGACAGCGGCCCG

CTGCTCATAAGACTC

GGCCTTAGAACCCCA

GTATCAGCAGAAGGA

CATTTTAGGACGGGA

CTTGGGTGACTCTAG

GGCACTGGTTTTCTT

TCCAGAGAGCGGAAC

AGGCGAGGAAAAGTA

GTCCCTTCTCGGCGA

TTCTGCGGAGGGATC

TCCGTGGGGCGGTGA

ACGCCGATGATTATA

TAAGGACGCGCCGGG

TGTGGCACAGCTAGT

TCCGTCGCAGCCGGG

ATTTGGGTCGCGGTT

CTTGTTTGTGGATCG

CTGTGATCGTCACTT

GGTGAGTTGCGGGCT

GCTGGGCTGGCCGGG

GCTTTCGTGGCCGCC

GGGCCGCTCGGTGGG

ACGGAAGCGTGTGGA

GAGACCGCCAAGGGC

TGTAGTCTGGGTCCG

CGAGCAAGGTTGCCC

TGAACTGGGGGTTGG

GGGGAGCGCACAAAA

TGGCGGCTGTTCCCG

AGTCTTGAATGGAAG

ACGCTTGTAAGGCGG

GCTGTGAGGTCGTTG

AAACAAGGTGGGGGG

CATGGTGGGCGGCAA

GAACCCAAGGTCTTG

AGGCCTTCGCTAATG

CGGGAAAGCTCTTAT

TCGGGTGAGATGGGC

TGGGGCACCATCTGG

GGACCCTGACGTGAA

GTTTGTCACTGACTG

GAGAACTCGGGTTTG

TCGTCTGGTTGCGGG

GGCGGCAGTTATGCG

GTGCCGTTGGGCAGT

GCACCCGTACCTTTG

GGAGCGCGCGCCTCG

TCGTGTCGTGACGTC

ACCCGTTCTGTTGGC

TTATAATGCAGGGTG

GGGCCACCTGCCGGT

AGGTGTGCGGTAGGC

TTTTCTCCGTCGCAG

GACGCAGGGTTCGGG

CCTAGGGTAGGCTCT

CCTGAATCGACAGGC

GCCGGACCTCTGGTG

AGGGGAGGGATAAGT

GAGGCGTCAGTTTCT

TTGGTCGGTTTTATG

TACCTATCTTCTTAA

GTAGCTGAAGCTCCG

GTTTTGAACTATGCG

CTCGGGGTTGGCGAG

TGTGTTTTGTGAAGT

TTTTTAGGCACCTTT

TGAAATGTAATCATT

TGGGTCAATATGTAA

TTTTCAGTGTTAGAC

TAGTAAA

50
SV40 Poly A
GTTTATTGCAGCTTA

TAATGGTTACAAATA

AAGCAATAGCATCAC

AACCAAACTCATCAA

TGTATCTTATCA

51
bHG Poly A
GACTGTGCCTTCTAG

TTGCCAGCCATCTGT

TGTTTGCCCCTCCCC

CGTGCCTTCCTTGAC

CCTGGAAGGTGCCAC

TCCCACTGTCCTTTC

CTAATAAAATGAGGA

AATTGCATCGCATTG

TCTGAGTAGGTGTCA

TTCTATTCTGGGGGG

TGGGGTGGGGCAGGA

CAGCAAGGGGGAGGA

TTGGGAAGACAATAG

CAGGCATGCTGGGGA

TGCGGTGGGCTCTAT

GG

52
RD114
ATGAAACTCCCAACA

Envelope
GGAATGGTCATTTTA

TGTAGCCTAATAATA

GTTCGGGCAGGGTTT

GACGACCCCCGCAAG

GCTATCGCATTAGTA

CAAAAACAACATGGT

AAACCATGCGAATGC

AGCGGAGGGCAGGTA

TCCGAGGCCCCACCG

AACTCCATCCAACAG

GTAACTTGCCCAGGC

AAGACGGCCTACTTA

ATGACCAACCAAAAA

TGGAAATGCAGAGTC

ACTCCAAAAAATCTC

ACCCCTAGCGGGGGA

GAACTCCAGAACTGC

CCCTGTAACACTTTC

CAGGACTCGATGCAC

AGTTCTTGTTATACT

GAATACCGGCAATGC

AGGGCGAATAATAAG

ACATACTACACGGCC

ACCTTGCTTAAAATA

CGGTCTGGGAGCCTC

AACGAGGTACAGATA

TTACAAAACCCCAAT

CAGCTCCTACAGTCC

CCTTGTAGGGGCTCT

ATAAATCAGCCCGTT

TGCTGGAGTGCCACA

GCCCCCATCCATATC

TCCGATGGTGGAGGA

CCCCTCGATACTAAG

AGAGTGTGGACAGTC

CAAAAAAGGCTAGAA

CAAATTCATAAGGCT

ATGCATCCTGAACTT

CAATACCACCCCTTA

GCCCTGCCCAAAGTC

AGAGATGACCTTAGC

CTTGATGCACGGACT

TTTGATATCCTGAAT

ACCACTTTTAGGTTA

CTCCAGATGTCCAAT

TTTAGCCTTGCCCAA

GATTGTTGGCTCTGT

TTAAAACTAGGTACC

CCTACCCCTCTTGCG

ATACCCACTCCCTCT

TTAACCTACTCCCTA

GCAGACTCCCTAGCG

AATGCCTCCTGTCAG

ATTATACCTCCCCTC

TTGGTTCAACCGATG

CAGTTCTCCAACTCG

TCCTGTTTATCTTCC

CCTTTCATTAACGAT

ACGGAACAAATAGAC

TTAGGTGCAGTCACC

TTTACTAACTGCACC

TCTGTAGCCAATGTC

AGTAGTCCTTTATGT

GCCCTAAACGGGTCA

GTCTTCCTCTGTGGA

AATAACATGGCATAC

ACCTATTTACCCCAA

AACTGGACAGGACTT

TGCGTCCAAGCCTCC

CTCCTCCCCGACATT

GACATCATCCCGGGG

GATGAGCCAGTCCCC

ATTCCTGCCATTGAT

CATTATATACATAGA

CCTAAACGAGCTGTA

CAGTTCATCCCTTTA

CTAGCTGGACTGGGA

ATCACCGCAGCATTC

ACCACCGGAGCTACA

GGCCTAGGTGTCTCC

GTCACCCAGTATACA

AAATTATCCCATCAG

TTAATATCTGATGTC

CAAGTCTTATCCGGT

ACCATACAAGATTTA

CAAGACCAGGTAGAC

TCGTTAGCTGAAGTA

GTTCTCCAAAATAGG

AGGGGACTGGACCTA

CTAACGGCAGAACAA

GGAGGAATTTGTTTA

GCCTTACAAGAAAAA

TGCTGTTTTTATGCT

AACAAGTCAGGAATT

GTGAGAAACAAAATA

AGAACCCTACAAGAA

GAATTACAAAAACGC

AGGGAAAGCCTGGCA

TCCAACCCTCTCTGG

ACCGGGCTGCAGGGC

TTTCTTCCGTACCTC

CTACCTCTCCTGGGA

CCCCTACTCACCCTC

CTACTCATACTAACC

ATTGGGCCATGCGTT

TTCAATCGATTGGTC

CAATTTGTTAAAGAC

AGGATCTCAGTGGTC

CAGGCTCTGGTTTTG

ACTCAGCAATATCAC

CAGCTAAAACCCATA

GAGTACGAGCCATGA

53
GALV
ATGCTTCTCACCTCA

Envelope
AGCCCGCACCACCTT

CGGCACCAGATGAGT

CCTGGGAGCTGGAAA

AGACTGATCATCCTC

TTAAGCTGCGTATTC

GGAGACGGCAAAACG

AGTCTGCAGAATAAG

AACCCCCACCAGCCT

GTGACCCTCACCTGG

CAGGTACTGTCCCAA

ACTGGGGACGTTGTC

TGGGACAAAAAGGCA

GTCCAGCCCCTTTGG

ACTTGGTGGCCCTCT

CTTACACCTGATGTA

TGTGCCCTGGCGGCC

GGTCTTGAGTCCTGG

GATATCCCGGGATCC

GATGTATCGTCCTCT

AAAAGAGTTAGACCT

CCTGATTCAGACTAT

ACTGCCGCTTATAAG

CAAATCACCTGGGGA

GCCATAGGGTGCAGC

TACCCTCGGGCTAGG

ACCAGGATGGCAAAT

TCCCCCTTCTACGTG

TGTCCCCGAGCTGGC

CGAACCCATTCAGAA

GCTAGGAGGTGTGGG

GGGCTAGAATCCCTA

TACTGTAAAGAATGG

AGTTGTGAGACCACG

GGTACCGTTTATTGG

CAACCCAAGTCCTCA

TGGGACCTCATAACT

GTAAAATGGGACCAA

AATGTGAAATGGGAG

CAAAAATTTCAAAAG

TGTGAACAAACCGGC

TGGTGTAACCCCCTC

AAGATAGACTTCACA

GAAAAAGGAAAACTC

TCCAGAGATTGGATA

ACGGAAAAAACCTGG

GAATTAAGGTTCTAT

GTATATGGACACCCA

GGCATACAGTTGACT

ATCCGCTTAGAGGTC

ACTAACATGCCGGTT

GTGGCAGTGGGCCCA

GACCCTGTCCTTGCG

GAACAGGGACCTCCT

AGCAAGCCCCTCACT

CTCCCTCTCTCCCCA

CGGAAAGCGCCGCCC

ACCCCTCTACCCCCG

GCGGCTAGTGAGCAA

ACCCCTGCGGTGCAT

GGAGAAACTGTTACC

CTAAACTCTCCGCCT

CCCACCAGTGGCGAC

CGACTCTTTGGCCTT

GTGCAGGGGGCCTTC

CTAACCTTGAATGCT

ACCAACCCAGGGGCC

ACTAAGTCTTGCTGG

CTCTGTTTGGGCATG

AGCCCCCCTTATTAT

GAAGGGATAGCCTCT

TCAGGAGAGGTCGCT

TATACCTCCAACCAT

ACCCGATGCCACTGG

GGGGCCCAAGGAAAG

CTTACCCTCACTGAG

GTCTCCGGACTCGGG

TCATGCATAGGGAAG

GTGCCTCTTACCCAT

CAACATCTTTGCAAC

CAGACCTTACCCATC

AATTCCTCTAAAAAC

CATCAGTATCTGCTC

CCCTCAAACCATAGC

TGGTGGGCCTGCAGC

ACTGGCCTCACCCCC

TGCCTCTCCACCTCA

GTTTTTAATCAGTCT

AAAGACTTCTGTGTC

CAGGTCCAGCTGATC

CCCCGCATCTATTAC

CATTCTGAAGAAACC

TTGTTACAAGCCTAT

GACAAATCACCCCCC

AGGTTTAAAAGAGAG

CCTGCCTCACTTACC

CTAGCTGTCTTCCTG

GGGTTAGGGATTGCG

GCAGGTATAGGTACT

GGCTCAACCGCCCTA

ATTAAAGGGCCCATA

GACCTCCAGCAAGGC

CTAACCAGCCTCCAA

ATCGCCATTGACGCT

GACCTCCGGGCCCTT

CAGGACTCAATCAGC

AAGCTAGAGGACTCA

CTGACTTCCCTATCT

GAGGTAGTACTCCAA

AATAGGAGAGGCCTT

GACTTACTATTCCTT

AAAGAAGGAGGCCTC

TGCGCGGCCCTAAAA

GAAGAGTGCTGTTTT

TATGTAGACCACTCA

GGTGCAGTACGAGAC

TCCATGAAAAAACTT

AAAGAAAGACTAGAT

AAAAGACAGTTAGAG

CGCCAGAAAAACCAA

AACTGGTATGAAGGG

TGGTTCAATAACTCC

CCTTGGTTTACTACC

CTACTATCAACCATC

GCTGGGCCCCTATTG

CTCCTCCTTTTGTTA

CTCACTCTTGGGCCC

TGCATCATCAATAAA

TTAATCCAATTCATC

AATGATAGGATAAGT

GCAGTCAAAATTTTA

GTCCTTAGACAGAAA

TATCAGACCCTAGAT

AACGAGGAAAACCTT

TAA

54
FUG
ATGGTTCCGCAGGTT

Envelope
CTTTTGTTTGTACTC

CTTCTGGGTTTTTCG

TTGTGTTTCGGGAAG

TTCCCCATTTACACG

ATACCAGACGAACTT

GGTCCCTGGAGCCCT

ATTGACATACACCAT

CTCAGCTGTCCAAAT

AACCTGGTTGTGGAG

GATGAAGGATGTACC

AACCTGTCCGAGTTC

TCCTACATGGAACTC

AAAGTGGGATACATC

TCAGCCATCAAAGTG

AACGGGTTCACTTGC

ACAGGTGTTGTGACA

GAGGCAGAGACCTAC

ACCAACTTTGTTGGT

TATGTCACAACCACA

TTCAAGAGAAAGCAT

TTCCGCCCCACCCCA

GACGCATGTAGAGCC

GCGTATAACTGGAAG

ATGGCCGGTGACCCC

AGATATGAAGAGTCC

CTACACAATCCATAC

CCCGACTACCACTGG

CTTCGAACTGTAAGA

ACCACCAAAGAGTCC

CTCATTATCATATCC

CCAAGTGTGACAGAT

TTGGACCCATATGAC

AAATCCCTTCACTCA

AGGGTCTTCCCTGGC

GGAAAGTGCTCAGGA

ATAACGGTGTCCTCT

ACCTACTGCTCAACT

AACCATGATTACACC

ATTTGGATGCCCGAG

AATCCGAGACCAAGG

ACACCTTGTGACATT

TTTACCAATAGCAGA

GGGAAGAGAGCATCC

AACGGGAACAAGACT

TGCGGCTTTGTGGAT

GAAAGAGGCCTGTAT

AAGTCTCTAAAAGGA

GCATGCAGGCTCAAG

TTATGTGGAGTTCTT

GGACTTAGACTTATG

GATGGAACATGGGTC

GCGATGCAAACATCA

GATGAGACCAAATGG

TGCCCTCCAGATCAG

TTGGTGAATTTGCAC

GACTTTCGCTCAGAC

GAGATCGAGCATCTC

GTTGTGGAGGAGTTA

GTTAAGAAAAGAGAG

GAATGTCTGGATGCA

TTAGAGTCCATCATG

ACCACCAAGTCAGTA

AGTTTCAGACGTCTC

AGTCACCTGAGAAAA

CTTGTCCCAGGGTTT

GGAAAAGCATATACC

ATATTCAACAAAACC

TTGATGGAGGCTGAT

GCTCACTACAAGTCA

GTCCGGACCTGGAAT

GAGATCATCCCCTCA

AAAGGGTGTTTGAAA

GTTGGAGGAAGGTGC

CATCCTCATGTGAAC

GGGGTGTTTTTCAAT

GGTATAATATTAGGG

CCTGACGACCATGTC

CTAATCCCAGAGATG

CAATCATCCCTCCTC

CAGCAACATATGGAG

TTGTTGGAATCTTCA

GTTATCCCCCTGATG

CACCCCCTGGCAGAC

CCTTCTACAGTTTTC

AAAGAAGGTGATGAG

GCTGAGGATTTTGTT

GAAGTTCACCTCCCC

GATGTGTACAAACAG

ATCTCAGGGGTTGAC

CTGGGTCTCCCGAAC

TGGGGAAAGTATGTA

TTGATGACTGCAGGG

GCCATGATTGGCCTG

GTGTTGATATTTTCC

CTAATGACATGGTGC

AGAGTTGGTATCCAT

CTTTGCATTAAATTA

AAGCACACCAAGAAA

AGACAGATTTATACA

GACATAGAGATGAAC

CGACTTGGAAAGTAA

55
LCMV
ATGGGTCAGATTGTG

Envelope
ACAATGTTTGAGGCT

CTGCCTCACATCATC

GATGAGGTGATCAAC

ATTGTCATTATTGTG

CTTATCGTGATCACG

GGTATCAAGGCTGTC

TACAATTTTGCCACC

TGTGGGATATTCGCA

TTGATCAGTTTCCTA

CTTCTGGCTGGCAGG

TCCTGTGGCATGTAC

GGTCTTAAGGGACCC

GACATTTACAAAGGA

GTTTACCAATTTAAG

TCAGTGGAGTTTGAT

ATGTCACATCTGAAC

CTGACCATGCCCAAC

GCATGTTCAGCCAAC

AACTCCCACCATTAC

ATCAGTATGGGGACT

TCTGGACTAGAATTG

ACCTTCACCAATGAT

TCCATCATCAGTCAC

AACTTTTGCAATCTG

ACCTCTGCCTTCAAC

AAAAAGACCTTTGAC

CACACACTCATGAGT

ATAGTTTCGAGCCTA

CACCTCAGTATCAGA

GGGAACTCCAACTAT

AAGGCAGTATCCTGC

GACTTCAACAATGGC

ATAACCATCCAATAC

AACTTGACATTCTCA

GATCGACAAAGTGCT

CAGAGCCAGTGTAGA

ACCTTCAGAGGTAGA

GTCCTAGATATGTTT

AGAACTGCCTTCGGG

GGGAAATACATGAGG

AGTGGCTGGGGCTGG

ACAGGCTCAGATGGC

AAGACCACCTGGTGT

AGCCAGACGAGTTAC

CAATACCTGATTATA

CAAAATAGAACCTGG

GAAAACCACTGCACA

TATGCAGGTCCTTTT

GGGATGTCCAGGATT

CTCCTTTCCCAAGAG

AAGACTAAGTTCTTC

ACTAGGAGACTAGCG

GGCACATTCACCTGG

ACTTTGTCAGACTCT

TCAGGGGTGGAGAAT

CCAGGTGGTTATTGC

CTGACCAAATGGATG

ATTCTTGCTGCAGAG

CTTAAGTGTTTCGGG

AACACAGCAGTTGCG

AAATGCAATGTAAAT

CATGATGCCGAATTC

TGTGACATGCTGCGA

CTAATTGACTACAAC

AAGGCTGCTTTGAGT

AAGTTCAAAGAGGAC

GTAGAATCTGCCTTG

CACTTATTCAAAACA

ACAGTGAATTCTTTG

ATTTCAGATCAACTA

CTGATGAGGAACCAC

TTGAGAGATCTGATG

GGGGTGCCATATTGC

AATTACTCAAAGTTT

TGGTACCTAGAACAT

GCAAAGACCGGCGAA

ACTAGTGTCCCCAAG

TGCTGGCTTGTCACC

AATGGTTCTTACTTA

AATGAGACCCACTTC

AGTGATCAAATCGAA

CAGGAAGCCGATAAC

ATGATTACAGAGATG

TTGAGGAAGGATTAC

ATAAAGAGGCAGGGG

AGTACCCCCCTAGCA

TTGATGGACCTTCTG

ATGTTTTCCACATCT

GCATATCTAGTCAGC

ATCTTCCTGCACCTT

GTCAAAATACCAACA

CACAGGCACATAAAA

GGTGGCTCATGTCCA

AAGCCACACCGATTA

ACCAACAAAGGAATT

TGTAGTTGTGGTGCA

TTTAAGGTGCCTGGT

GTAAAAACCGTCTGG

AAAAGACGCTGA

56
FPV Envelope
ATGAACACTCAAATC

CTGGTTTTCGCCCTT

GTGGCAGTCATCCCC

ACAAATGCAGACAAA

ATTTGTCTTGGACAT

CATGCTGTATCAAAT

GGCACCAAAGTAAAC

ACACTCACTGAGAGA

GGAGTAGAAGTTGTC

AATGCAACGGAAACA

GTGGAGCGGACAAAC

ATCCCCAAAATTTGC

TCAAAAGGGAAAAGA

ACCACTGATCTTGGC

CAATGCGGACTGTTA

GGGACCA

TTACCGGACCACCTC

AATGCGACCAATTTC

TAGAATTTTCAGCTG

ATCTAATAATCGAGA

GACGAGAAGGAAATG

ATGTTTGTTACCCGG

GGAAGTTTGTTAATG

AAGAGGCATTGCGAC

AAATCCTCAGAGGAT

CAGGTGGGATTGACA

AAGAAACAATGGGAT

TCACATATAGTGGAA

TAAGGACCAACGGAA

CAACTAGTGCATGTA

GAAGATCAGGGTCTT

CATTCTATGCAGAAA

TGGAGTGGCTCCTGT

CAAATACAGACAATG

CTGCTTTCCCACAAA

TGACAAAATCATACA

AAAACACAAGGAGAG

AATCAGCTCTGATAG

TCTGGGGAATCCACC

ATTCAGGATCAACCA

CCGAACAGACCAAAC

TATATGGGAGTGGAA

ATAAACTGATAACAG

TCGGGAGTTCCAAAT

ATCATCAATCTTTTG

TGCCGAGTCCAGGAA

CACGACCGCAGATAA

ATGGCCAGTCCGGAC

GGATTGATTTTCATT

GGTTGATCTTGGATC

CCAATGATACAGTTA

CTTTTAGTTTCAATG

GGGCTTTCATAGCTC

CAAATCGTGCCAGCT

TCTTGAGGGGAAAGT

CCATGGGGATCCAGA

GCGATGTGCAGGTTG

ATGCCAATTGCGAAG

GGGAATGCTACCACA

GTGGAGGGACTATAA

CAAGCAGATTGCCTT

TTCAAAACATCAATA

GCAGAGCAGTTGGCA

AATGCCCAAGATATG

TAAAACAGGAAAGTT

TATTATTGGCAACTG

GGATGAAGAACGTTC

CCGAACCTTCCAAAA

AAAGGAAAAAAAGAG

GCCTGTTTGGCGCTA

TAGCAGGGTTTATTG

AAAATGGTTGGGAAG

GTCTGGTCGACGGGT

GGTACGGTTTCAGGC

ATCAGAATGCACAAG

GAGAAGGAACTGCAG

CAGACTACAAAAGCA

CCCAATCGGCAATTG

ATCAGATAACCGGAA

AGTTAAATAGACTCA

TTGAGAAAACCAACC

AGCAATTTGAGCTAA

TAGATAATGAATTCA

CTGAGGTGGAAAAGC

AGATTGGCAATTTAA

TTAACTGGACCAAAG

ACTCCATCACAGAAG

TATGGTCTTACAATG

CTGAACTTCTTGTGG

CAATGGAAAACCAGC

ACACTATTGATTTGG

CTGATTCAGAGATGA

ACAAGCTGTATGAGC

GAGTGAGGAAACAAT

TAAGGGAAAATGCTG

AAGAGGATGGCACTG

GTTGCTTTGAAATTT

TTCATAAATGTGACG

ATGATTGTATGGCTA

GTATAAGGAACAATA

CTTATGATCACAGCA

AATACAGAGAAGAAG

CGATGCAAAATAGAA

TACAAATTGACCCAG

TCAAATTGAGTAGTG

GCTACAAAGATGTGA

TACTTTGGTTTAGCT

TCGGGGCATCATGCT

TTTTGCTTCTTGCCA

TTGCAATGGGCCTTG

TTTTCATATGTGTGA

AGAACGGAAACATGC

GGTGCACTATTTGTA

TATAA

57
RRV
AGTGTAACAGAGCAC

Envelope
TTTAATGTGTATAAG

GCTACTAGACCATAC

CTAGCACATTGCGCC

GATTGCGGGGACGGG

TACTTCTGCTATAGC

CCAGTTGCTATCGAG

GAGATCCGAGATGAG

GCGTCTGATGGCATG

CTTAAGATCCAAGTC

TCCGCCCAAATAGGT

CTGGACAAGGCAGGC

ACCCACGCCCACACG

AAGCTCCGATATATG

GCTGGTCATGATGTT

CAGGAATCTAAGAGA

GATTCCTTGAGGGTG

TACACGTCCGCAGCG

TGCTCCATACATGGG

ACGATGGGACACTTC

ATCGTCGCACACTGT

CCACCAGGCGACTAC

CTCAAGGTTTCGTTC

GAGGACGCAGATTCG

CACGTGAAGGCATGT

AAGGTCCAATACAAG

CACAATCCATTGCCG

GTGGGTAGAGAGAAG

TTCGTGGTTAGACCA

CACTTTGGCGTAGAG

CTGCCATGCACCTCA

TACCAGCTGACAACG

GCTCCCACCGACGAG

GAGATTGACATGCAT

ACACCGCCAGATATA

CCGGATCGCACCCTG

CTATCACAGACGGCG

GGCAACGTCAAAATA

ACAGCAGGCGGCAGG

ACTATCAGGTACAAC

TGTACCTGCGGCCGT

GACAACGTAGGCACT

ACCAGTACTGACAAG

ACCATCAACACATGC

AAGATTGACCAATGC

CATGCTGCCGTCACC

AGCCATGACAAATGG

CAATTTACCTCTCCA

TTTGTTCCCAGGGCT

GATCAGACAGCTAGG

AAAGGCAAGGTACAC

GTTCCGTTCCCTCTG

ACTAACGTCACCTGC

CGAGTGCCGTTGGCT

CGAGCGCCGGATGCC

ACCTATGGTAAGAAG

GAGGTGACCCTGAGA

TTACACCCAGATCAT

CCGACGCTCTTCTCC

TATAGGAGTTTAGGA

GCCGAACCGCACCCG

TACGAGGAATGGGTT

GACAAGTTCTCTGAG

CGCATCATCCCAGTG

ACGGAAGAAGGGATT

GAGTACCAGTGGGGC

AACAACCCGCCGGTC

TGCCTGTGGGCGCAA

CTGACGACCGAGGGC

AAACCCCATGGCTGG

CCACATGAAATCATT

CAGTACTATTATGGA

CTATACCCCGCCGCC

ACTATTGCCGCAGTA

TCCGGGGCGAGTCTG

ATGGCCCTCCTAACT

CTGGCGGCCACATGC

TGCATGCTGGCCACC

GCGAGGAGAAAGTGC

CTAACACCGTACGCC

CTGACGCCAGGAGCG

GTGGTACCGTTGACA

CTGGGGCTGCTTTGC

TGCGCACCGAGGGCG

AATGCA

58
MLV 10A1
ATGGAAGGTCCAGCG

Envelope
TTCTCAAAACCCCTT

AAAGATAAGATTAAC

CCGTGGAAGTCCTTA

ATGGTCATGGGGGTC

TATTTAAGAGTAGGG

ATGGCAGAGAGCCCC

CATCAGGTCTTTAAT

GTAACCTGGAGAGTC

ACCAACCTGATGACT

GGGCGTACCGCCAAT

GCCACCTCCCTTTTA

GGAACTGTACAAGAT

GCCTTCCCAAGATTA

TATTTTGATCTATGT

GATCTGGTCGGAGAA

GAGTGGGACCCTTCA

GACCAGGAACCATAT

GTCGGGTATGGCTGC

AAATACCCCGGAGGG

AGAAAGCGGACCCGG

ACTTTTGACTTTTAC

GTGTGCCCTGGGCAT

ACCGTAAAATCGGGG

TGTGGGGGGCCAAGA

GAGGGCTACTGTGGT

GAATGGGGTTGTGAA

ACCACCGGACAGGCT

TACTGGAAGCCCACA

TCATCATGGGACCTA

ATCTCCCTTAAGCGC

GGTAACACCCCCTGG

GACACGGGATGCTCC

AAAATGGCTTGTGGC

CCCTGCTACGACCTC

TCCAAAGTATCCAAT

TCCTTCCAAGGGGCT

ACTCGAGGGGGCAGA

TGCAACCCTCTAGTC

CTAGAATTCACTGAT

GCAGGAAAAAAGGCT

AATTGGGACGGGCCC

AAATCGTGGGGACTG

AGACTGTACCGGACA

GGAACAGATCCTATT

ACCATGTTCTCCCTG

ACCCGCCAGGTCCTC

AATATAGGGCCCCGC

ATCCCCATTGGGC

CTAATCCCGTGATCA

CTGGTCAACTACCCC

CCTCCCGACCCGTGC

AGATCAGGCTCCCCA

GGCCTCCTCAGCCTC

CTCCTACAGGCGCAG

CCTCTATAGTCCCTG

AGACTGCCCCACCTT

CTCAACAACCTGGGA

CGGGAGACAGGCTGC

TAAACCTGGTAGAAG

GAGCCTATCAGGCGC

TTAACCTCACCAATC

CCGACAAGACCCAAG

AATGTTGGCTGTGCT

TAGTGTCGGGACCTC

CTTATTACGAAGGAG

TAGCGGTCGTGGGCA

CTTATACCAATCATT

CTACCGCCCCGGCCA

GCTGTACGGCCACTT

CCCAACATAAGCTTA

CCCTATCTGAAGTGA

CAGGACAGGGC

CTATGCATGGGAGCA

CTACCTAAAACTCAC

CAGGCCTTATGTAAC

ACCACCCAAAGTGCC

GGCTCAGGATCCTAC

TACCTTGCAGCACCC

GCTGGAACAATGTGG

GCTTGTAGCACTGGA

TTGACTCCCTGCTTG

TCCACCACGATGCTC

AATCTAACCACAGAC

TATTGTGTATTAGTT

GAGCTCTGGCCCAGA

ATAATTTACCACTCC

CCCGATTATATGTAT

GGTCAGCTTGAACAG

CGTACCAAATATAAG

AGGGAGCCAGTATCG

TTGACCCTGGCCCTT

CTGCTAGGAGGATTA

ACCATGGGAGGGATT

GCAGCTGGAATAGGG

ACGGGGACCACTGCC

CTAATCAAAACCCAG

CAGTTTGAGCAGCTT

CACGCCGCTATCCAG

ACAGACCTCAACGAA

GTCGAAAAATCAATT

ACCAACCTAGAAAAG

TCACTGACCTCGTTG

TCTGAAGTAGTCCTA

CAGAACCGAAGAGGC

CTAGATTTGCTCTTC

CTAAAAGAGGGAGGT

CTCTGCGCAGCCCTA

AAAGAAGAATGTTGT

TTTTATGCAGACCAC

ACGGGACTAGTGAGA

GACAGCATGGCCAAA

CTAAGGGAAAGGCTT

AATCAGAGACAAAAA

CTATTTGAGTCAGGC

CAAGGTTGGTTCGAA

GGGCAGTTTAATAGA

TCCCCCTGGTTTACC

ACCTTAATCTCCACC

ATCATGGGACCTCTA

ATAGTACTCTTACTG

ATCTTACTCTTTGGA

CCCTGCATTCTCAAT

CGATTGGTCCAATTT

GTTAAAGACAGGATC

TCAGTGGTCCAGGCT

CTGGTTTTGACTCAA

CAATATCACCAGCTA

AAACCTATAGAGTAC

GAGCCATGA

59
EboV
ATGGGTGTTACAGGA

Envelope
ATATTGCAGTTACCT

CGTGATCGATTCAAG

AGGACATCATTCTTT

CTTTGGGTAATTATC

CTTTTCCAAAGAACA

TTTTCCATCCCACTT

GGAGTCATCCACAAT

AGCACATTACAGGTT

AGTGATGTCGACAAA

CTGGTTTGCCGTGAC

AAACTGTCATCCACA

AATCAATTGAGATCA

GTTGGACTGAATCTC

GAAGGGAATGGAGTG

GCAACTGACGTGCCA

TCTGCAACTAAAAGA

TGGGGCTTCAGGTCC

GGTGTCCCACCAAAG

GTGGTCAATTATGAA

GCTGGTGAATGGGCT

GAAAACTGCTACAAT

CTTGAAATCAAAAAA

CCTGACGGGAGTGAG

TGTCTACCAGCAGCG

CCAGACGGGATTCGG

GGCTTCCCCCGGTGC

CGGTATGTGCACAAA

GTATCAGGAACGGGA

CCGTGTGCCGGAGAC

TTTGCCTTCCACAAA

GAGGGTGCTTTCTTC

CTGTATGACCGACTT

GCTTCCACAGTTATC

TACCGAGGAACGACT

TTCGCTGAAGGTGTC

GTTGCATTTCTGATA

CTGCCCCAAGCTAAG

AAGGACTTCTTCAGC

TCACACCCCTTGAGA

GAGCCGGTCAATGCA

ACGGAGGACCCGTCT

AGTGGCTACTATTCT

ACCACAATTAGATAT

CAAGCTACCGGTTTT

GGAACCAATGAGACA

GAGTATTTGTTCGAG

GTTGACAATTTGACC

TACGTCCAACTTGAA

TCAAGATTCACACCA

CAGTTTCTGCTCCAG

CTGAATGAGACAATA

TATACAAGTGGGAAA

AGGAGCAATACCACG

GGAAAACTAATTTGG

AAGGTCAACCCCGAA

ATTGATACAACAATC

GGGGAGTGGGCCTTC

TGGGAAACTAAAAAA

ACCTCACTAGAAAAA

TTCGCAGTGAAGAGT

TGTCTTTCACAGCTG

TATCAAACAGAGCCA

AAAACATCAGTGGTC

AGAGTCCGGCGCGAA

CTTCTTCCGACCCAG

GGACCAACACAACAA

CTGAAGACCACAAAA

TCATGGCTTCAGAAA

ATTCCTCTGCAATGG

TTCAAGTGCACAGTC

AAGGAAGGGAAGCTG

CAGTGTCGCATCTGA

CAACCCTTGCCACAA

TCTCCACGAGTCCTC

AACCCCCCACAACCA

AACCAGGTCCGGACA

ACAGCACCCACAATA

CACCCGTGTATAAAC

TTGACATCTCTGAGG

CAACTCAAGTTGAAC

AACATCACCGCAGAA

CAGACAACGACAGCA

CAGCCTCCGACACTC

CCCCCGCCACGACCG

CAGCCGGACCCCTAA

AAGCAGAGAACACCA

ACACGAGCAAGGGTA

CCGACCTCCTGGACC

CCGCCACCACAACAA

GTCCCCAAAACCACA

GCGAGACCGCTGGCA

ACAACAACACTCATC

ACCAAGATACCGGAG

AAGAGAGTGCCAGCA

GCGGGAAGCTAGGCT

TAATTACCAATACTA

TTGCTGGAGTCGCAG

GACTGATCACAGGCG

GGAGGAGAGCTCGAA

GAGAAGCAATTGTCA

ATGCTCAACCCAAAT

GCAACCCTAATTTAC

ATTACTGGACTACTC

AGGATGAAGGTGCTG

CAATCGGACTGGCCT

GGATACCATATTTCG

GGCCAGCAGCCGAGG

GAATTTACATAGAGG

GGCTGATGCACAATC

AAGATGGTTTAATCT

GTGGGTTGAGACAGC

TGGCCAACGAGACGA

CTCAAGCTCTTCAAC

TGTTCCTGAGAGCCA

CAACCGAGCTACGCA

CCTTTTCAATCCTCA

ACCGTAAGGCAATTG

ATTTCTTGCTGCAGC

GATGGGGCGGCACAT

GCCACATTTTGGGAC

CGGACTGCTGTATCG

AACCACATGATTGGA

CCAAGAACATAACAG

ACAAAATTGATCAGA

TTATTCATGATTTTG

TTGATAAAACCCTTC

CGGACCAGGGGGACA

ATGACAATTGGTGGA

CAGGATGGAGACAAT

GGATACCGGCAGGTA

TTGGAGTTACAGGCG

TTATAATTGCAGTTA

TCGCTTTATTCTGTA

TATGCAAATTTGTCT

TTTAG

60
Thyroxin
CTTTCTCTTTTGTTT

binding
TACATGAAGGGTCTG

globulin
GCAGCCAAAGCAATC

promoter
ACTCAAAGTTCAAAC

(TBG)
CTTATCATTTTTTGC

TTTGTTCCTCTTGGC

CTTGGTTTTGTACAT

CAGCTTTGAAAATAC

CATCCCAGGGTTAAT

GCTGGGGTTAATTTA

TAACTAAGAGTGCTC

TAGTTTTGCAATACA

GGACATGCTATAAAA

ATGGAAAGATGTTGC

TTTCTGAG

61
DNA
GCGAGAACTTGTGCC

fragment
TCCCCGTGTTCCTGC

containing
TCTTTGTCCCTCTGT

prothrombin
CCTACTTAGACTAAT

enhancer and
ATTTGCCTTGGGTAC

human alpha-1
TGCAAACAGGAAATG

anti-trypsin
GGGGAGGGACAGGAG

promoter
TAGGGCGGAGGGTAG

CCCGGGGATCTTGCT

ACCAGTGGAACAGCC

ACTAAGGATTCTGCA

GTGAGAGCAGAGGGC

CAGCTAAGTGGTACT

CTCCCAGAGACTGTC

TGACTCACGCCACCC

CCTCCACCTTGGACA

CAGGACGCTGTGGTT

TCTGAGCCAGGTACA

ATGACTCCTTTCGGT

AAGTGCAGTGGAAGC

TGTACACTGCCCAGG

CAAAGCGTCCGGGCA

GCGTAGGCGGGCGAC

TCAGATCCCAGCCAG

TGGACTTAGCCCCTG

TTTGCTCCTCCGATA

ACTGGGGTGACCTTG

GTTAATATTCACCAG

CAGCCTCCCCCGTTG

CCCCTCTGGATCCAC

TGCTTAAATACGGAC

GAGGACAGGGCCCTG

TCTCCTCAGCTTCAG

GCACCACCACTGACC

TGGGACAGTGAAT

62
DNA
GTTAATCATTAACGT

fragment
TAATCATTAACGTTA

containing
ATCATTAACGTTAAT

prothrombin
CATTAACGTTAATCA

enhancer,
TTAACATCGATGCGA

human alpha-1
GAACTTGTGCCTCCC

anti-trypsin
CGTGTTCCTGCTCTT

promoter, and
TGTCCCTCTGTCCTA

five HNF1
CTTAGACTAATATTT

binding sites
GCCTTGGGTACTGCA

AACAGGAAATGGGGG

AGGGACAGGAGTAGG

GCGGAGGGTAGGATT

CTGCAGTGAGAGCAG

AGGGCCAGCTAAGTG

GTACTCTCCCAGAGA

CTGTCTGACTCACGC

CACCCCCTCCACCTT

GGACACAGGACGCTG

TGGTTTCTGAGCCAG

GTACAATGACTCCTT

TCGGTAAGTGCAGTG

GAAGCTGTACACTGC

CCAGGCAAAGCGTCC

GGGCAGCGTAGGCGG

GCGACTCAGATCCCA

GCCAGTGGACTTAGC

CCCTGTTTGCTCCTC

CGATAACTGGGGTGA

CCTTGGTTAATATTC

ACCAGCAGCCTCCCC

CGTTGCCCCTCTGGA

TCCACTGCTTAAATA

CGGACGAGGACAGGG

CCCTGTCTCCTCAGC

TTCAGGCACCACCAC

TGACCTGGGACAGTG

AAT

63
DNA
GTTAATCATTAACGC

fragment
TTGTACTTTGGTACA

containing
GTTAATCATTAACGC

prothrombin
TTGTACTTTGGTACA

enhancer,
GTTAATCATTAACGC

human alpha-1
TTGTACTTTGGTACA

anti-trypsin
ATCGATGCGAGAACT

promoter, and
TGTGCCTCCCCGTGT

three
TCCTGCTCTTTGTCC

HNF1/HNF4
CTCTGTCCTACTTAG

binding sites
ACTAATATTTGCCTT

GGGTACTGCAAACAG

GAAATGGGGGAGGGA

CAGGAGTAGGGCGGA

GGGTAGCCCGGGGAT

TCTGCAGTGAGAGCA

GAGGGCCAGCTAAGT

GGTACTCTCCCAGAG

ACTGTCTGACTCACG

CCACCCCCTCCACCT

TGGACACAGGACGCT

GTGGTTTCTGAGCCA

GGTACAATGACTCCT

TTCGGTAAGTGCAGT

GGAAGCTGTACACTG

CCCAGGCAAAGCGTC

CGGGCAGCGTAGGCG

GGCGACTCAGATCCC

AGCCAGTGGACTTAG

CCCCTGTTTGCTCCT

CCGATAACTGGGGTG

ACCTTGGTTAATATT

CACCAGCAGCCTCCC

CCGTTGCCCCTCTGG

ATCCACTGCTTAAAT

ACGGACGAGGACAGG

GCCCTGTCTCCTCAG

CTTCAGGCACCACCA

CTGACCTGGGACAGT

GAAT

64
hPAH FAM
TCGTGAAAGCTCATG

TaqMan
GACAGTGGC

Probe

65
PAH TaqMan
AGATCTTGAGGCATG

Forward
ACATTGG

Primer

66
PAH TaqMan
GTCCAGCTCTTGAAT

Reverse
GGTTCTT

Primer

67
Actin FAM
AGCGGGAAATCGTGC

Probe
GTGAC

68
Actin Forward
GGACCTGACTGACTA

Primer
CCTCAT

69
Actin Reverse
CGTAGCACAGCTTCT

Primer
CCTTAAT

70
Codon-
ATGTCTACCGCCGTG

optimized
CTGGAAAATCCTGGC

PAH (OPT3)
CTGGGCAGAAAGCTG

AGCGACTTCGGCCAA

GAGACAAGCTACATC

GAGGACAACTGCAAC

CAGAACGGCGCCATC

AGCCTGATCTTCAGC

CTGAAAGAAGAAGTG

GGCGCCCTGGCCAAG

GTGCTGAGACTGTTC

GAAGAGAACGACGTG

AACCTGACACACATC

GAGAGCAGACCCAGC

AGACTGAAGAAGGAC

GAGTACGAGTTCTTC

ACCCACCTGGACAAG

CGGAGCCTGCCTGCT

CTGACCAACATCATC

AAGATCCTGCGGCAC

GACATCGGCGCCACA

GTGCACGAACTGAGC

CGGGACAAGAAAAAG

GACACCGTGCCATGG

TTCCCCAGAACCATC

CAAGAGCTGGACAGA

TTCGCCAACCAGATC

CTGAGCTATGGCGCC

GAGCTGGACGCTGAT

CACCCTGGCTTTAAG

GACCCCGTGTACCGG

GCCAGAAGAAAGCAG

TTTGCCGATATCGCC

TACAACTACCGG

CACGGCCAGCCTATT

CCTCGGGTCGAGTAC

ATGGAAGAGGAAAAG

AAAACCTGGGGCACC

GTGTTCAAGACCCTG

AAGTCCCTGTACAAG

ACCCACGCCTGCTAC

GAGTACAACCACATC

TTCCCACTGCTCGAG

AAGTACTGCGGCTTC

CACGAGGACAATATC

CCTCAGCTCGAGGAC

GTGTCCCAGTTCCTG

CAGACCTGCACCGGC

TTTAGACTGAGGCCT

GTTGCCGGACTGCTG

AGCAGCAGAGATTTT

CTCGGCGGCCTGGCC

TTCAGAGTGTTCCAC

TGTACCCAGTACATC

AGACACGGCAGCAAG

CCCATGTACACCCCT

GAGCCTGATATCTGC

CACGAGCTGCTGGGA

CATGTGCCCCTGTTC

AGCGATAGAAGCTTC

GCCCAGTTCAGCCAA

GAGATCGGACTGGCT

TCTCTGGGAGCCCCT

GACGAGTACATTGAG

AAGCTGGCCACCATC

TACTGGTTCACCGTG

GAGTTCGGCCTGTGC

AAGCAGGGCGATAGC

ATCAAGGCTTATGGC

GCTGGCCTGCTGTCT

AGCTTTGGCGAGCTG

CAGTACTGTCTGAGC

GAGAAGCCTAAGCTG

CTGCCCCTGGAACTG

GAAAAGACCGCCATC

CAGAACTACACCGTG

ACCGAGTTCCAGCCT

CTGTACTACGTGGCC

GAGAGCTTCAACGAC

GCCAAAGAAAAAGTG

CGGAACTTCGCCGCC

ACCATTCCTCGGCCT

TTCAGCGTCAGATAC

GACCCCTACACACAG

CGGATCGAGGTGCTG

GACAACACACAGCAG

CTGAAAATTCTGGCC

GACAGCATCAACAGC

GAGATCGGCATCCTG

TGCAGCGCCCTGCAG

AAAATCAAGTGA

71
Codon-
ATGAGTACGGCTGTG

optimized
CTCGAGAATCCAGGT

PAH
TTGGGCCGAAAGCTG

(OPT2/3)
TCTGATTTTGGACAG

GAGACATCTTATATT

GAAGACAACTGCAAC

CAGAATGGTGCGATA

TCCCTTATTTTTTCT

CTGAAAGAAGAAGTA

GGTGCGCTGGCAAAG

GTCTTGCGGCTGTTT

GAAGAGAACGATGTT

AATCTTACTCATATT

GAGTCCAGACCATCA

CGGCTGAAAAAAGAC

GAGTACGATCATTAA

GATCCTCCGGCATGA

CATAGGGGCGACAGT

GCATGAGCTTTCAAG

GGATAAAAAGAAAGA

TACCGTCCCCTGGTT

TCCAAGGACCATACA

AGAACTCGACCGATT

CGCGAACCAGATCCT

TTCATATGGTGCTGA

GTTGGATGCTGACCA

CCCCGGCTTCAAAGA

CCCGGTCTACCGAGC

GCGGCGGAAACAATT

TGCTGACATCGCATA

CAATTACAGGCATGG

CCAGCCAATTCCTAG

AGTAGAATACATGGA

AGAAGAGAAAAAAAC

CTGGGGTACCGTCTT

CAAGACGCTGAAATC

ATTGTATAAAACTCA

TGCATGTTACGAATA

TAACCATATTTTTCC

GTTGCTCGAGAAATA

TTGCGGGTTCCACGA

AGATAACATCCCACA

ACTCGAGGATGTATC

TCAGTTCCTCCAGAC

CTGTACGGGGTTTCG

ACTTAGGCCTGTTGC

CGGACTGCTGAGCAG

CAGAGATTTTCTCGG

CGGCCTGGCCTTCAG

AGTGTTCCACTGTAC

CCAGTACATCAGACA

CGGCAGCAAGCCCAT

GTACACCCCTGAGCC

TGATATCTGCCACGA

GCTGCTGGGACATGT

GCCCCTGTTCAGCGA

TAGAAGCTTCGCCCA

GTTCAGCCAAGAGAT

CGGACTGGCTTCTCT

GGGAGCCCCTGACGA

GTACATTGAGAAGCT

GGCCACCATCTACTG

GTTCACCGTGGAGTT

CGGCCTGTGCAAGCA

GGGCGATAGCATCAA

GGCTTATGGCGCTGG

CCTGCTGTCTAGCTT

TGGCGAGCTGCAGTA

CTGTCTGAGCGAGAA

GCCTAAGCTGCTGCC

CCTGGAACTGGAAAA

GACCGCCATCCAGAA

CTACACCGTGACCGA

GTTCCAGCCTCTGTA

CTACGTGGCCGAGAG

CTTCAACGACGCCAA

AGAAAAAGTGCGGAA

CTTCGCCGCCACCAT

TCCTCGGCCTTTCAG

CGTCAGATACGACCC

CTACACACAGCGGAT

CGAGGTGCTGGACAA

CACACAGCAGCTGAA

AATTCTGGCCGACAG

CATCAACAGCGAGAT

CGGCATCCTGTGCAG

CGCCCTGCAGAAAAT

CAAGTGA

72
Codon-
ATGTCTACCGCCGTG

optimized
CTGGAAAATCCTGGC

PAH
CTGGGCAGAAAGCTG

(OPT3/2)
AGCGACTTCGGCCAA

GAGACAAGCTACATC

GAGGACAACTGCAAC

CAGAACGGCGCCATC

AGCCTGATCTTCAGC

CTGAAAGAAGAAGTG

GGCGCCCTGGCCAAG

GTGCTGAGACTGTTC

GAAGAGAACGACGTG

AACC

TGACACACATCGAGA

GCAGACCCAGCAGAC

TGAAGAAGGACGAGT

ACGAGTTCTTCACCC

ACCTGGACAAGCGGA

GCCTGCCTGCTCTGA

CCAACATCATCAAGA

TCCTGCGGCACGACA

TCGGCGCCACAGTGC

ACGAACTGAGCCGGG

ACAAGAAAAAGGACA

CCGTGCCATGGTTCC

CCAGAACCATCCAAG

AGCTGGACAGATTCG

CCAACCAGATCCTGA

GCTATGGCGCCGAGC

TGGACGCTGATCACC

CTGGCTTTAAGGACC

CCGTGTACCGGGCCA

GAAGAAAGCAGTTTG

CCGATATCGCCTACA

ACTACCGGCACGGCC

AGCCTATTCCTCGGG

TCGAGTACATGGAAG

AGGAAAAGAAAACCT

GGGGCACCGTGTTCA

AGACCCTGAAGTCCC

TGTACAAGACCCACG

CCTGCTACGAGTACA

ACCACATCTTCCCAC

TGCTCGAGAAGTACT

GCGGCTTCCACGAGG

ACAATATCCCTCAGC

TCGAGGACGTGTCCC

AGTTCCTGCAGACCT

GCACCGGCTTTAGAC

TGAGGCCTGTCGCGG

GTTTGCTCAGTTCTC

GAGACTTCCTGGGTG

GATTGGCGTTTCGGG

TATTCCATTGCACGC

AGTATATCCGACACG

GAAGTAAGCCAATGT

ACACGCCAGAACCCG

ATATCTGTCACGAAT

TGCTTGGACACGTTC

CTCTGTTTTCTGATC

GATCATTCGCTCAGT

TTTCACAGGAAATCG

GCCTGGCATCTTTGG

GAGCGCCGGATGAAT

ATATTGAGAAGCTCG

CTACAATTTACTGGT

TCACGGTAGAATTTG

GGTTGTGCAAGCAGG

GTGATAGTATTAAAG

CATACGGTGCGGGAT

TGCTGTCCTCATTCG

GGGAGCTTCAGTATT

GCCTGTCCGAGAAAC

CCAAGCTGTTGCCGT

TGGAATTGGAAAAAA

CCGCTATCCAAAATT

ACACAGTAACGGAGT

TCCAACCTTTGTACT

ACGTAGCCGAGTCAT

TTAACGATGCAAAGG

AGAAGGTCAGAAATT

TTGCTGCGACGATAC

CCAGACCGTTCTCAG

TAAGGTACGATCCTT

ACACTCAGAGGATTG

AAGTCCTGGATAATA

CGCAACAGCTCAAGA

TCCTGGCAGACTCCA

TAAATTCTGAAATCG

GCATCTTGTGTTCAG

CACTGCAAAAGATAA

AATAA

73
DNA
AGAACCATCCAAGAG

Fragment of

OPT3

74
DNA
TATTCCTCGGGTCGA

Fragment of
GTAC

OPT3

75
DNA
AGAGATCGGACTGGC

Fragment of
T

OPT3

76
DNA
TCCTCGGCCTTTCAG

Fragment of

OPT3

77
DNA
GTTAATCATTAACGC

fragment
TTGTACTTTGGTACA

containing
ATCGATGCGAGAACT

prothrombin
TGTGCCTCCCCGTGT

enhancer,
TCCTGCTCTTTGTCC

human alpha-
CTCTGTCCTACTTAG

1, anti-trypsin
ACTAATATTTGCCTT

promoter,
GGGTACTGCAAACAG

and
GAAATGGGGGAGGGA

one
CAGGAGTAGGGCGGA

HNF1/HNF4
GGGTAGCCCGGGGAT

binding
TCTGCAGTGAGAGCA

site
GAGGGCCAGCTAAGT

GGTACTCTCCCAGAG

ACTGTCTGACTCACG

CCACCCCCTCCACCT

TGGACACAGGACGCT

GTGGTTTCTGAGCCA

GGTACAATGACTCCT

TTCGGTAAGTGCAGT

GGAAGCTGTACACTG

CCCAGGCAAAGCGTC

CGGGCAGCGTAGGCG

GGCGACTCAGATCCC

AGCCAGTGGACTTAG

CCCCTGTTTGCTCCT

CCGATAACTGGGGTG

ACCTTGGTTAATATT

CACCAGCAGCCTCCC

CCGTTGCCCCTCTGG

ATCCACTGCTTAAAT

ACGGACGAGGACAGG

GCCCTGTCTCCTCAG

CTTCAGGCACCACCA

CTGACCTGGGACAGT

GAAT

78
Prothrombin
GCGAGAACTTGTGCC

enhancer-
TCCCCGTGTTCCTGC

hAAT
TCTTTGTCCCTCTGT

promoter-
CCTACTTAGACTAAT

ATTTGCCTTGGGTAC

TGCAAACAGGAAATG

GGGGAGGGACAGGAG

TAGGGCGGAGGGTAG

CCCGGGGATCTTGCT

ACCAGTGGAACAGCC

ACTAAGGATTCTGCA

GTGAGAGCAGAGGGC

CAGCTA

Minute Virus
AGTGGTACTCTCCCA

of Mouse
GAGACTGTCTGACTC

intron
ACGCCACCCCCTCCA

CCTTGGACACAGGAC

GCTGTGGTTTCTGAG

CCAGGTACAATGACT

CCTTTCGGTAAGTGC

AGTGGAAGCTGTACA

CTGCCCAGGCAAAGC

GTCCGGGCAGCGTAG

GCGGGCGACTCAGAT

CCCAGCCAGTGGACT

TAGCCCCTGTTTGCT

CCTCCGATAACTGGG

GTGACCTTGGTTAAT

ATTCACCAGCAGCCT

CCCCCGTTGCCCCTC

TGGATCCACTGCTTA

AATACGGACGAGGAC

AGGGCCCTGTCTCCT

CAGCTTCAGGCACCA

CCACTGACCTGGGAC

AGTGAATAAGAGGTA

AGGGTTTAAGGGATG

GTTGGTTGGTGGGGT

ATTAATGTTTAATTA

CCTGGAGCACCTGCC

TGAAATCACTTTTTT

TCAGGTTGG

79
hAAT
GGGGGAGGCTGCTGG

promoter-
TGAATATTAACCAAG

Transthyretin
GTCACCCCAGTTATC

enhancer-
GGAGGAGCAAACAGG

Minute
GGCTAAGTCCACCGA

Virus
TGCTCTAATCTCTCT

of Mouse
AGACAAGGTTCATAT

intron
TTGTATGGGTTACTT

ATTCTCTCTTTGTTG

ACTAAGTCAATAATC

AGAATCAGCAGGTTT

GCAGTCAGATTGGCA

GGGATAAGCAGCCTA

GCTCAGGAGAAGTGA

GTATAAAAGCCCCAG

GCTGGGAGCAGCCAT

CAAAGAGGTAAGGGT

TTAAGGGATGGTTGG

TTGGTGGGGTATTAA

TGTTTAATTACCTGG

AGCACCTGCCTGAAA

TCACTTTTTTTCAGG

TTGG

80
Minute
AAGAGGTAAGGGTTT

virus
AAGGGATGGTTGGTT

of Mouse
GGTGGGGTATTAATG

intron
TTTAATTACCTGGAG

CACCTGCCTGAAATC

ACTTTTTTTCAGGTT

GG

81
Transthyretin
CCGATGCTCTAATCT

enhancer
CTCTAGACAAGGTTC

ATATTTGTATGGGTT

ACTTATTCTCTCTTT

GTTGACTAAGTCAAT

AATCAGAATCAGCAG

GTTTGCAGTCAGATT

GGCAGGGATAAGCAG

CCTAGCTCAGGAGAA

GTGAGTATAAAAGCC

CCAGGCTGGGAGCAG

CCATCA

82
hAAT
GGGGGAGGCTGCTGG

promoter
TGAATATTAACCAAG

GTCACCCCAGTTATC

GGAGGAGCAAACAGG

GGCTAAGTCCA

83
PAH
ATGTCTACCGCCGTG

optimized
CTGGAAAATCCTGGC

version
CTGGGCAGAAAGCTG

3-PAH
AGCGACTTCGGCCAA

3′UTR
GAGACAAGCTACATC

GAGGACAACTGCAAC

CAGAACGGCGCCATC

AGCCTGATCTTCAGC

CTGAAAGAAGAAGTG

GGCGCCCTGGCCAAG

GTGCTGAGACTGTTC

GAAGAGAACGACGTG

AACCTGACACACATC

GAGAGCAGACCCAGC

AGACTGAAGAAGGAC

GAGTACGAGTTCTTC

ACCCACCTGGACAAG

CGGAGCCTGCCTGCT

CTGACCAACATCATC

AAGATCCTGCGGCAC

GACATCGGCGCCACA

GTGCACGAACTGAGC

CGGGACAAGAAAAAG

GACACCGTGCCATGG

TTCCCCAGAACCATC

CAAGAGCTGGACAGA

TTCGCCAACCAGATC

CTGAGCTATGGCGCC

GAGCTGGACGCTGAT

CACCCTGGCTTTAAG

GACCCCGTGTACCGG

GCCAGAAGAAAGCAG

TTTGCCGATATCGCC

TACAACTACCGGCAC

GGCCAGCCTATTCCT

CGGGTCGAGTACATG

GAAGAGGAAAAGAAA

ACCTGGGGCACCGTG

TTCAAGACCCTGAAG

TCCCTGTACAAGACC

CACGCCTGCTACGAG

TACAACCACATCTTC

CCACTGCTCGAGAAG

TACTGCGGCTTCCAC

GAGGACAATATCCCT

CAGCTCGAGGACGTG

TCCCAGTTCCTGCAG

ACCTGCACCGGCTTT

AGACTGAGGCCTGTT

GCCGGACTGCTGAGC

AGCAGAGATTTTCTC

GGCGGCCTGGCCTTC

AGAGTGTTCCACTGT

ACCCAGTACATCAGA

CACGGCAGCAAGCCC

ATGTACACCCCTGAG

CCTGATATCTGCCAC

GAGCTGCTGGGACAT

GTGCCCCTGTTCAGC

GATAGAAGCTTCGCC

CAGTTCAGCCAAGAG

ATCGGACTGGCTTCT

CTGGGAGCCCCTGAC

GAGTACATTGAGAAG

CTGGCCACCATCTAC

TGGTTCACCGTGGAG

TTCGGCCTGTGCAAG

CAGGGCGATAGCATC

AAGGCTTATGGCGCT

GGCCTGCTGTCTAGC

TTTGGCGAGCTGCAG

TACTGTCTGAGCGAG

AAGCCTAAGCTGCTG

CCCCTGGAACTGGAA

AAGACCGCCATCCAG

AACTACACCGTGACC

GAGTTCCAGCCTCTG

TACTACGTGGCCGAG

AGCTTCAACGACGCC

AAAGAAAAAGTGCGG

AACTT

CGCCGCCACCATTCC

TCGGCCTTTCAGCGT

CAGATACGACCCCTA

CACACAGCGGATCGA

GGTGCTGGACAACAC

ACAGCAGCTGAAAAT

TCTGGCCGACAGCAT

CAACAGCGAGATCGG

CATCCTGTGCAGCGC

CCTGCAGAAAATCAA

GTGAGTCGACAGCCA

TGGACAGAATGTGGT

CTGTCAGCTGTGAAT

CTGTTGATGGAGATC

CAACTATTTCTTTCA

TCAGAAAAAGTCCGA

AAAGCAAACCTTAAT

TTGAAATAACAGCCT

TAAATCCTTTACAAG

ATGGAGAAACAACAA

ATAAGTCAAAATAAT

CTGAAATGACAGGAT

ATGAGTACATACTCA

AGAGCATAATGGTAA

ATCTTTTGGGGTCAT

CTTTGATTTAGAGAT

GATAATCCCATACTC

TCAATTGAGTTAAAT

CAGTAATCTGTCGCA

TTTCATCAAGATTA

84
PAH
ATGTCTACCGCCGTG

optimized
CTGGAAAATCCTGGC

version 3-
CTGGGCAGAAAGCTG

Albumin
AGCGACTTCGGCCAA

3′UTR
GAGACAAGCTACATC

GAGGACAACTGCAAC

CAGAACGGCGCCATC

AGCCTGATCTTCAGC

CTGAAAGAAGAAGTG

GGCGCCCTGGCCAAG

GTGCTGAGACTGTTC

GAAGAGAACGACGTG

AACCTGACACACATC

GAGAGCAGACCCAGC

AGACTGAAGAAGGAC

GAGTACGAGTTCTTC

ACCCACCTGGACAAG

CGGAGCCTGCCTGCT

CTGACCAACATCATC

AAGATCCTGCGGCAC

GACATCGGCGCCACA

GTGCACGAACTGAGC

CGGGACAAGAAAAAG

GACACCGTGCCATGG

TTCCCCAGAACCATC

CAAGAGCTGGACAGA

TTCGCCAACCAGATC

CTGAGCTATGGCGCC

GAGCTGGACGCTGAT

CACCCTGGCTTTAAG

GACCCCGTGTACCGG

GCCAGAAGAAAGCAG

TTTGCCGATATCGCC

TACAACTACCGGCAC

GGCCAGCCTATTCCT

CGGGTCGAGTACATG

GAAGAGGAAAAGAAA

ACCTGGGGCACCGTG

TTCAAGACCCTGAAG

TCCCTGTACAAGACC

CACGCCTGCTACGAG

TACAACCACATCTTC

CCACTGCTCGAGAAG

TACTGCGGCTTCCAC

GAGGACAATATCCCT

CAGCTCGAGGACGTG

TCCCAGTTCCTGCAG

ACCTGCACCGGCTTT

AGACTGAGGCCTGTT

GCCGGACTGCTGAGC

AGCAGAGATTTTCTC

GGCGGCCTGGCCTTC

AGAGTGTTCCACTGT

ACCCAGTACATCAGA

CACGGCAGCAAGCCC

ATGTACACCCCTGAG

CCTGATATCTGCCAC

GAGCTGCTGGGACAT

GTGCCCCTGTTCAGC

GATAGAAGCTTCGCC

CAGTTCAGCCAAGAG

ATCGGACTGGCTTCT

CTGGGAGCCCCTGAC

GAGTACATTGAGAAG

CTGGCCACCATCTAC

TGGTTCACCGTGGAG

TTCGGCCTGTGCAAG

CAGGGCGATAGCATC

AAGGCTTATGGCGCT

GGCCTGCTGTCTAGC

TTTGGCGAGCTGCAG

TACTGTCTGAGCGAG

AAGCCTAAGCTGCTG

CCCCTGGAACTGGAA

AAGACCGCCATCCAG

AACTACACCGTGACC

GAGTTCCAGCCTCTG

TACTACGTGGCCGAG

AGCTTCAACGACGCC

AAAGAAAAAGTGCGG

AACTTCGCCGCCACC

ATTCCTCGGCCTTTC

AGCGTCAGATACGAC

CCCTACACACAGCGG

ATCGAGGTGCTGGAC

AACACACAGCAGCTG

AAAATTCTGGCCGAC

AGCATCAACAGCGAG

ATCGGCATCCTGTGC

AGCGCCCTGCAGAAA

ATCAAGTGAGTCGAC

ATTCAGCAGCCGTAA

GTCTAGGACAGGCTT

AAATTGTTTTCACTG

GTGTAAATTGCAGAA

AGATGATCTAAGTAA

TTTGGCATTTATTTT

AATAGGTTTGAAAAA

CACATGCCATTTTAC

AAATAAGACTTATAT

TTGTCCTTTTGTTTT

TCAGCCTACCATGAG

AATAAGAGAAAGAAA

ATGAAGATCAAAAGC

TTATTCATCTGTTTT

TCTTTTTCGTTGGTG

TAAAGCCAACACCCT

GTCTAAAAAACATAA

ATTTCTTTAATCATT

TTGCCTCTTTTCTCT

GTGCTTCAATTAATA

AAAAATGGAAAGAAT

CTAATAGAGTGGTAC

AGCACTGTTATTTTT

CAAAGATGTGTTGCT

ATCCTGAAAATTCTG

TAGGTTCTGTGGAAG

TTCCAGTGTTCTCTC

TTATTCCACTTCGGT

AGAGGATTTCTAGTT

TCTTGTGGGCTAATT

AAATAAATCATTAAT

ACTCTTCTAAGTTAT

GGATTATAAACATTC

AAAATAATATTTTGA

CATTATGATAATTCT

GAATAAAAGAACAAA

AACCATGGTATAGGT

AAGGAATATAAAACA

TGGCTTTTACCTTAG

AAAAAACAATTCTAA

AATTCATATGGAATC

AAAAAAGAGCCTGCA

85
PAH 3′UTR
AGCCATGGACAGAAT

GTGGTCTGTCAGCTG

TGAATCTGTTGATGG

AGATCCAACTATTTC

TTTCATCAGAAAAAG

TCCGAAAAGCAAACC

TTAATTTGAAATAAC

AGCCTTAAATCCTTT

ACAAGATGGAGAAAC

AACAAATAAGTCAAA

ATAATCTGAAATGAC

AGGATATGAGTACAT

ACTCAAGAGCATAAT

GGTAAATCTTTTGGG

GTCATCTTTGATTTA

GAGATGATAATCCCA

TACTCTCAATTGAGT

86
Albumin
ATTCAGCAGCCGTAA

3′UTR
GTCTAGGACAGGCTT

AAATTGTTTTCACTG

GTGTAAATTGCAGAA

AGATGATCTAAGTAA

TTTGGCATTTATTTT

AATAGGTTTGAAAAA

CACATGCCATTTTAC

AAATAAGACTTATAT

TTGTCCTTTTGTTTT

TCAGCCTACCATGAG

AATAAGAGAAAGAAA

ATGAAGATCAAAAGC

TTATTCATCTGTTTT

TCTTTTTCGTTGGTG

TAAAGCCAACACCCT

GTCTAAAAAACATAA

ATTTCTTTAATCATT

TTGCCTCTTTTCTCT

GTGCTTCAATTAATA

AAAAATGGAAAGAAT

CTAATAGAGTGGTAC

AGCACTGTTATTTTT

CAAAGATGTGTTGCT

ATCCTGAAAATTCTG

TAGGTTCTGTGGAAG

TTCCAGTGTTCTCTC

TTATTCCACTTCGGT

AGAGGATTTCTAGTT

TCTTGTGGGCTAATT

AAATAAATCATTAAT

ACTCTTCTAAGTTAT

GGATTATAAACATTC

AAAATAATATTTTGA

CATTATGATAATTCT

GAATAAAAGAACAAA

AACCATGGTATAGGT

AAGGAATATAAAACA

TGGCTTTTACCTTAG

AAAAAACAATTCTAA

AATTCATATGGAATC

AAAAAAGAGCCTGCA

87
WPREs
AATCAACCTCTGGAT

(WPRE
TACAAAATTTGTGAA

without X-
AGATTGACTGATATT

protein
CTTAACTATGTTGCT

sequence)
CCTTTTACGCTGTGT

GGATATGCTGCTTTA

ATGCCTCTGTATCAT

GCTATTGCTTCCCGT

ACGGCTTTCGTTTTC

TCCTCCTTGTATAAA

TCCTGGTTGCTGTCT

CTTTATGAGGAGTTG

TGGCCCGTTGTCCGT

CAACGTGGCGTGGTG

TGCTCTGTGTTTGCT

GACGCAACCCCCACT

GGCTGGGGCATTGCC

ACCACCTGTCAACTC

CTTTCTGGGACTTTC

GCTTTCCCCCTCCCG

ATCGCCACGGCAGAA

CTCATCGCCGCCTGC

CTTGCCCGCTGCTGG

ACAGGGGCTAGGTTG

CTGGGCACTGATAAT

TCCGTGGTGTTGTCG

GTACC

OPTIMIZED PHENYLANANINE HYDROXYLASE EXPRESSION

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

PRIORITY AND INCORPORATION BY REFERENCE

PCT Information

Provisional Applications (1)