AAV-based sfasl gene therapy for the treatment of glaucoma

Abstract

Aspects of the disclosure relate to compositions and methods for preventing optic nerve degeneration. In some embodiments, compositions and methods provided herein are useful for treating glaucoma. The disclosure relates, in part, to the discovery that a combination of codon-optimization of soluble Fas ligand (sFasL)-encoding transgenes and certain "MCI" capsid proteins (e.g., rAAVs encoding codon-optimized sFasL encapsidated by MCI capsid proteins) improves delivery and expression of sFasL peptides to the eye of a subject and prevents death of retinal ganglion cells (RGCs) and axons.

Description

[0001] U0120.70214WQ00

[0002] AAV-BASED SFASL GENE THERAPY FOR THE TREATMENT OF GLAUCOMA RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 63 / 754,786, filed February 6, 2025, and entitled “AAV-BASED SFASL GENE THERAPY FOR THE TREATMENT OF GLAUCOMA,” and to U.S. Provisional Patent Application Serial No. 63 / 787,046, filed April 11, 2025, and entitled “AAV-BASED SFASL GENE THERAPY FOR THE TREATMENT OF GLAUCOMA,” the entire contents of each of which are herein incorporated by reference in their entirety for all purposes.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (U012070214WO00-SEQ-KZM.xml; Size: 70,394 bytes; and Date of Creation: February 4, 2026) are herein incorporated by reference in their entirety.

[0006] BACKGROUND

[0007] Glaucoma, a leading cause of blindness worldwide, is a complex multifactorial disease characterized by the progressive loss of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is a well-recognized risk factor for the development of glaucoma and remains the only modifiable disease-associated parameter. However, reduction of IOP alone does not prevent loss of RGCs in all patients and RGC destruction can continue even after IOP has been successfully lowered. Furthermore, a high incidence of glaucoma with loss of RGCs occurs in patients having normal IOP.

[0008] SUMMARY

[0009] Aspects of the disclosure relate to compositions and methods for preventing optic nerve degeneration. In some embodiments, compositions and methods provided herein are useful for treating glaucoma. The disclosure relates, in part, to the discovery that a combination of codon-optimization of soluble Fas ligand (sFasL)-encoding transgenes and certain “MCI” capsid proteins (e.g., rAAVs encoding codon-optimized sFasL encapsidated

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[0011] by MCI capsid proteins) improves delivery and expression of sFasL peptides to the eye of a subject and prevents death of retinal ganglion cells (RGCs) and axons. In some embodiments, compositions described herein prevent ocular diseases (e.g., glaucoma) prior to onset of symptoms. In some embodiments, compositions described herein reduce or ameliorate pathology associated with certain ocular diseases (e.g., glaucoma) after the onset of symptoms.

[0012] Accordingly, in some aspects, the disclosure provides an isolated nucleic acid comprising a transgene flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), the transgene comprising a promoter operably linked to a nucleic acid sequence encoding a codon-optimized soluble Fas Ligand (sFasL) peptide. Soluble Fas Ligandencoding nucleic acid sequences are generally, known, for example as described in International Application Number PCT / US2017 / 040735, filed July 5, 2017, and published as W02018 / 009553 on January 11, 2018; the entire contents of which are incorporated by reference herein.

[0013] In some embodiments, a codon-optimized sFasL peptide is a mammalian sFasL peptide. In some embodiments, a codon-optimized sFasL peptide is a canine sFasL peptide, a human sFasL peptide, or a mouse sFasL peptide.

[0014] In some embodiments, a sFasL peptide comprises the amino acid sequence in any one of SEQ ID NOs: 1-3.

[0015] In some embodiments, a nucleic acid sequence encoding a canine sFasL peptide comprises a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, or 99% identical to the sequence set forth in any one of SEQ ID NOs: 4-6. In some embodiments, a nucleic acid sequence encoding a canine sFasL peptide comprises the sequence set forth in any one of SEQ ID NOs: 4-6.

[0016] In some embodiments, a nucleic acid sequence encoding a human sFasL peptide comprises a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, or 99% identical to the sequence set forth in any one of SEQ ID NOs: 7-9. In some embodiments, a nucleic acid sequence encoding a human sFasL peptide comprises the sequence set forth in any one of SEQ ID NOs: 7-9.

[0017] In some embodiments, a nucleic acid sequence encoding a mouse sFasL peptide comprises a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, or 99% identical

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[0019] to the sequence set forth in any one of SEQ ID NOs: 10-12. In some embodiments, a nucleic acid sequence encoding a mouse sFasL peptide comprises the sequence set forth in any one of SEQ ID NOs: 10-12.

[0020] In some embodiments, a promoter comprises a constitutive promoter, inducible promoter, or tissue-specific promoter. In some embodiments a promoter comprises a chicken beta-actin (CBA) promoter.

[0021] In some embodiments, AAV ITRs are AAV2 ITRs. In some embodiments, one of the AAV ITRs is a mutant ITR.

[0022] In some embodiments, an isolated nucleic acid described herein is located on a plasmid. In some embodiments, the plasmid comprises a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, or 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 13-21. In some embodiments, the plasmid comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 13-21.

[0023] In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising an isolated nucleic acid encoding a codon-optimized sFasL peptide, and one or more AAV capsid proteins.

[0024] In some embodiments, one or more capsid proteins are AAV2 capsid proteins, or variants thereof. In some embodiments, an AAV2 capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a variant of the AAV2 capsid protein comprises the following amino acid substitutions relative to SEQ ID NO: 22: E36G, D80N, and V125A. In some embodiments, one or more AAV capsid proteins comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0025] In some embodiments, one or more AAV capsid proteins are MCI capsid proteins (e.g., AAC2.MC1, AAV3.MC1, AAV5.MC1, etc.). In some embodiments, an MCI capsid protein comprises a glycine (G) at position 36, an asparagine (N) at position 80, and an alanine (A) at position 125 with reference to amino acid position numbering of a wild-type AAV2 capsid protein. MCI capsid proteins are known, for example as described in International Application No. PCT / US2024 / 024867, filed April 17, 2024, and published as WO2024 / 220462 on October 24, 2024; the entire contents of which are incorporated by reference herein.

[0026] - 3 - #14792861vlIn some aspects, the disclosure provides a method for treating or preventing an ocular disease or disorder in a subject, the method comprising administering an isolated nucleic acid as described herein, or an rAAV as described herein, to a subject in need thereof. In some embodiments, a subject is a mammal. In some embodiments, subject is a human, canine, or mouse.

[0027] In some embodiments, the amount of sFasL or a fragment thereof is effective in reducing glaucoma disease progression, lowering intraocular pressure in the subject, inactivating retinal glial cells, inhibiting TNFoc activity, reducing retinal ganglion cell (RGC) death, and / or reducing axonal degeneration. In some embodiments, novel compositions and methods are provided for preventing optic nerve degeneration and / or for treating glaucoma.

[0028] In another aspect, the present disclosure provides methods for treating a Fas liganddependent inflammatory condition. In some embodiments, the Fas ligand-dependent inflammatory condition is glaucoma or cutaneous lupus.

[0029] In some embodiments, any of the subjects to be treated by methods described herein may have been treated with another glaucoma therapy (e.g., eyedrops, oral medications, or surgery). In some embodiments, methods described herein may further comprise administering to the subject another anti-glaucoma treatment.

[0030] Also provided in the disclosure are (a) pharmaceutical compositions for use treating glaucoma in a subject. In some embodiments, such pharmaceutical compositions comprise one or more rAAVs that comprise a nucleic acid engineered to express sFasL or a fragment thereof described herein and a pharmaceutically acceptable carrier. In some embodiments, uses of rAAVs that comprise a nucleic acid engineered to express sFasL or a fragment thereof in manufacturing a medicament for glaucoma treatment are also provided.

[0031] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram of membrane-bound FasL (mFasL) and soluble FasL (sFasL).

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[0034] FIGs. 2A-2C are images that correspond to a demonstration of sFasL inhibitory activity. FIG. 2A depicts a schematic diagram of a plurality of FasL types. FIG. 2B is a plot depicting cytotoxicity of the different FasL types of FIG. 2A. FIG. 2C depicts a plurality of images indicating inflammation associated with the different FasL types of FIG. 2A.

[0035] FIGs. 3A-3B depict a transition from sFasL to mFasL in glaucoma in a microbead-induced model of glaucoma (B6 WT mice). FIG. 3 A depicts fluorescence intensity of mFasL, sFasL, and actin 0 days and 21 days post microbead. FIG. 3B is a plot of FasL / actin ratio vs. days post microbead for sFasL and mFasL.

[0036] FIG. 4A depicts DBA / 2J - chronic mouse model. At 2 months of age, an intravitreal injection AAV2. sFasL or AAV2.eGFP (3 x 109vg / 1 pl inj.) was conducted, and at 10 months of age, an RGC / axon analysis was conducted.

[0037] FIG. 4B is a plot of IOP vs. age for the chronic mouse model.

[0038] FIG. 4C is a plot of RGC cell density / mm2retina vs. group, wherein the groups are as follows: D2-Gp: no glaucoma; D2: Untreated DBA / 2J mice; D2-eGFP: DBA / 2J mice + AAV2-eGFP; and D2-sFasL: DBA / 2J mice + AAV2-sFasL.

[0039] FIG. 5A is a plot for a DBA / 2J chronic mouse model. At 7 months of age, an intravitreal injection (3 x 109PFU / 1 pl inj.) was conducted, and at 15 months of age, RGC / axon analysis was conducted.

[0040] FIG. 5B illustrates two plots: (1) RGC cell density / mm2vs. experimental group, and (2) axon density ( 104) / mm2vs. experimental group.

[0041] FIG. 6A is a plot of IOP vs. days post microbead injection for a microbead - inducible model.

[0042] FIG. 6B is a plot of amplitude vs. condition (four conditions), as an electrophysiological assessment of RGC function.

[0043] FIG. 7A is a fundic image revealing that tagging was strongest in the superior nasal region.

[0044] FIG. 7B is a light microscope image of a cross-section of FIG. 7A indicating high expression throughout the retinal layers from an outer nerve fiber layer to a retinal pigment epithelium layer.

[0045] FIG. 8A is a fundic image from 6 weeks post-injection revealing very strong expression in a superior nasal region.

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[0047] FIG. 8B is a light microscope image of a cross-section of FIG. 8A indicating inner nuclear layer tagging, nerve fiber layer, and inner plexiform layer.

[0048] FIG. 9 A is a fundic image showing GFP expression around the optic nerve head. FIG. 9B is a light microscope image of a cross-section of FIG. 9A showing little expression and one spot of tagging in inner nuclear layer.

[0049] FIG. 10 shows representative data indicating treatment of subjects with AAV-sFasL after disease onset halts disease progression.

[0050] FIG. 11 shows representative data from an AAV2-sFasL Pilot dog study. Briefly, delivery of 1011vg / eye led to good expression but was associated with uveitis, retinitis, and optic neuritis. Lowering the AAV-sFasL dose to IO10and 109vg / eye reduced inflammation but also reduced sFasL expression and was not neuroprotective.

[0051] FIG. 12 shows representative data indicating MCI AAV capsid protein mediates improved transduction and reduced immunogenicity when compared to AAV2 and AAV2.7m8 capsid-containing AAVs.

[0052] FIG. 13A shows representative data indicating that intraocular pressure increased in microbead (MB) injected mice.

[0053] FIG. 13B shows representative data indicating loss of ganglion cells in untreated control mice but not in mice injected with AAV-sFasL constructs or canine sFasL constructs.

[0054] DETAILED DESCRIPTION

[0055] Aspects of the disclosure relate to compositions and methods for preventing optic nerve degeneration. The disclosure relates, in part, to the discovery that a combination of codon-optimization of soluble Fas ligand (sFasL)-encoding transgenes and certain “MCI” capsid proteins (e.g., rAAVs encoding codon-optimized sFasL encapsidated by MCI capsid proteins) improves delivery and expression of sFasL peptides to the eye of a subject and prevents death of retinal ganglion cells (RGCs) and axons. In some embodiments, compositions and methods provided herein are useful for treating glaucoma.

[0056] Isolated nucleic acids

[0057] The disclosure relates, in some aspects, to isolated nucleic acids encoding a soluble FasL protein or a variant thereof. Fas Ligand (FasL) is a 40 kDa type II transmembrane

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[0059] protein of the TNF family, originally identified by its capacity to induce apoptosis in Fas receptor positive cells. FasL can be expressed as a membrane-bound protein (mFasL) or cleaved and released as a soluble protein (sFasL). The human FasL gene encodes for a 281 amino acid protein. In some embodiments, canine sFasL protein comprises the amino acid sequence set forth as SEQ ID NO: 1. In some embodiments, human sFasL protein comprises an amino acid sequence corresponding to amino acid residues 127-281 of human FasL. In some embodiments, human sFasL protein comprises the amino acid sequence set forth as SEQ ID NO: 2. In some embodiments, mouse sFasL protein comprises the amino acid sequence set forth as SEQ ID NO: 3.

[0060] Aspects of the disclosure relate to codon-optimized nucleic acid sequences. In some embodiments, the nucleic acid sequence encoding sFasL protein (e.g., human sFasL, canine sFasL, mouse sFasL, etc.) is a codon-optimized sequence (e.g., codon optimized for expression in mammalian cells). Without wishing to be bound by any particular theory, codon-optimization enables the reduction of certain undesirable characteristics in nucleic acid sequences, for example structural elements that may be immunogenic in a mammalian host (e.g., CpG islands, high GC content, etc.). In some embodiments, a codon-optimized sequence encoding sFasL protein comprises reduced GC content relative to a wild-type sequence that has not been codon-optimized. In some embodiments, a codon-optimized sequence encoding sFasL protein comprises a 1-5%, 3-5%, 3-10%, 5-10%, 5-15%, 10-20%, 15-30%, 20-40%, 25-50%, or 30-60% reduction in GC content relative to a wild-type sequence that has not been codon-optimized. In some embodiments, a codon-optimized sequence encoding sFasL protein comprises fewer guanine and / or cytosine nucleobases relative to a wild-type sequence that has not been codon-optimized. In some embodiments, a codon-optimized sequence encoding sFasL protein comprises 1-5, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer guanine and / or cytosine nucleobases relative to a wild- type sequence that has not been codon-optimized. In some embodiments, a codon-optimized sequence encoding sFasL protein comprises fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon-optimized. In some embodiments, a codon-optimized sequence encoding sFasL protein comprises 1-3, 3-5, 3-10, 5-10, 5-15, 10-20, 15-30, 20-40, 25-50, or 30-60 fewer CpG dinucleotide islands relative to a wild-type sequence that has not been codon-optimized. In some embodiments, a codon-optimized

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[0062] sequence encoding sFasL has been codon-optimized to improve replication and / or packaging of viral vectors (e.g., rAAVs) encoding the sFasL protein.

[0063] The level of identity or similarity of a codon-optimized sequence encoding a sFasL protein may vary with respect to a wild-type sFasL encoding sequence (e.g., canine sFasL wild-type sequence, human sFasL wild-type sequence, mouse sFasL wild-type sequence, etc.). In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL protein comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL protein comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL protein comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL protein comprises up to 20 nucleotides that are different from the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL protein comprises up to 20 nucleotides that are different from the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL protein comprises up to 20 nucleotides that are different from the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, a codon-optimized nucleic acid encoding sFasL protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more (e.g., 30, 40, 50, 100, 200, etc.) nucleotides that are different from the nucleic acid set forth in SEQ ID NO: 1. In some embodiments, a codon-optimized nucleic acid encoding sFasL protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more (e.g., 30, 40, 50, 100, 200, etc.) nucleotides that are different from the nucleic acid set forth in SEQ ID NO: 2. In some embodiments, a codon-optimized nucleic acid encoding sFasL protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more (e.g., 30, 40, 50, 100, 200, etc.) nucleotides that are different from the nucleic acid set forth in SEQ ID NO: 3.

[0064] In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100%

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[0066] identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 4-12. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 12.

[0067] In some aspects, the disclosure relates to codon-optimized nucleic acid sequences which result in increased sFasL expression in cells (e.g., mammalian cells) relative to sFasL expression by wild-type sFasL encoding constructs. Expression of sFasL by isolated nucleic acids and constructs described herein may be increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more (e.g., at least 20-fold, 50-fold, 100-fold, etc. relative to expression in by wild-type sFasL-encoding constructs.

[0068] A "nucleic acid" sequence refers to a DNA or RNA sequence. In some embodiments, proteins and nucleic acids of the disclosure are isolated. As used herein, the term “isolated” means artificially produced. As used herein with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered

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[0070] isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins or peptides, the term “isolated” refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0071] As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissuespecific, are known in the art and may be utilized.

[0072] As used herein, a nucleic acid sequence (e.g., coding sequence) and regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5’ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript

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[0074] might be translated into the desired protein or polypeptide. Similarly two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame. In some embodiments, operably linked coding sequences yield a fusion protein. In some embodiments, operably linked coding sequences yield a functional RNA (e.g., miRNA).

[0075] A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

[0076] For nucleic acids encoding proteins, a poly adenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence. A rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence would be used to produce a protein that contain more than one polypeptide chains. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al., and references cited therein at, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, a Foot and Mouth Disease Virus 2A sequence is included in polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal,

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[0078] 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

[0079] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen], In some embodiments, a promoter is an enhanced chicken P-actin promoter. In some embodiments, a promoter is a U6 promoter. In some embodiments, a promoter is a chicken beta-actin (CBA) promoter. In some embodiments, a promoter is a CB6 promoter.

[0080] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0081] In another embodiment, the native promoter for the transgene will be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic

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[0083] the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0084] In some embodiments, the regulatory sequences impart tissue- specific gene expression capabilities. In some cases, the tissue- specific regulatory sequences bind tissuespecific transcription factors that induce transcription in a tissue specific manner. Such tissuespecific regulatory sequences (e.g., promoters, enhancers, etc..) are well known in the art. In some embodiments, the tissue-specific promoter is an eye-specific promoter. Examples of eye-specific promoters include but are not limited to a retinoschisin promoter, K12 promoter, a rhodopsin promoter, a rod-specific promoter, a cone-specific promoter, a rhodopsin kinase promoter, a GRK1 promoter, an interphotoreceptor retinoid-binding protein proximal (IRBP) promoter, and an opsin promoter (e.g., a red opsin promoter, a blue opsin promoter, etc.).

[0085] rAAV Vectors

[0086] The isolated nucleic acids of the invention may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise, as disclosed elsewhere herein, one or more regions that encode one or more proteins (e.g., human sFasL, canine sFasL, mouse sFasE, or a fragment thereof). The transgene may also comprise a region encoding, for example, a miRNA binding site, and / or an expression control sequence (e.g., a poly-A tail), as described elsewhere in the disclosure.

[0087] Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is

[0088] - 13 - #14792861vlwithin the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K.

[0089] Fisher et al., J Virol., 70:520532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the isolated nucleic acid e.g., the rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR.

[0090] In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In some embodiments, the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS). The term “lacking a terminal resolution site” can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR). Without wishing to be bound by any particular theory, a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16( 10): 1648- 1656.

[0091] In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 13-21. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 14. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding

[0092] - 14 - #14792861vlU0120.70214WQ00

[0093] sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, an rAAV vector comprising a codon-optimized nucleic acid sequence encoding sFasL comprises the nucleotide sequence set forth in SEQ ID NO: 21.

[0094] In addition to the major elements identified above for the recombinant AAV vector, the vector also includes conventional control elements which are operably linked with elements of the transgene in a manner that permits its transcription, translation and / or expression in a cell transfected with the vector or infected with the virus produced by the invention.

[0095] Recombinant adeno-associated viruses (rAAVs)

[0096] In some aspects, the disclosure provides isolated AAVs. As used herein with respect to AAVs, the term “isolated” refers to an AAV that has been artificially produced or obtained. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs”. Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s). The AAV capsid is an important element in determining these tissue-specific targeting capabilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected.

[0097] Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003 / 0138772), the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a

[0098] - 15 - #14792861vlU0120.70214WQ00

[0099] host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. A A Vs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner.

[0100] The disclosure is based, in part, on the recognition that rAAVs comprising codon-optimized nucleic acids encoding sFasL and certain variants of AAV2 capsid protein and other capsid proteins, are both efficiently packaged in production cells (e.g., HEK293 cells) and delivered to ocular cells (e.g., retinal cells, photoreceptor cells, etc.) of a subject. In some embodiments, the AAV2 capsid protein variants are MCI capsid protein variants. MCI capsid proteins are known, for example as described in International Application No.

[0101] PCT / US2024 / 024867, filed April 17, 2024, and published as WO2024 / 220462 on October 24, 2024; the entire contents of which are incorporated by reference herein.

[0102] MCI capsid proteins generally refers to an AAV capsid protein (e.g., AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAV9, and AAV10) which comprises one or more amino acid substitutions selected from glycine (G) at position 36, asparagine (N) at position 80, and / or alanine (A) at position 125 corresponding to a wild-type AAV2 capsid protein (e.g., SEQ ID NO: 22). In some embodiments, an MCI capsid protein comprises mutations at positions corresponding to E36, D80, and V125 of SEQ ID NO: 2. In some embodiments, the mutations are E36G, D80N, and V125A. In some embodiments, an MCI capsid protein comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the MCI capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0103] - 16 - #14792861vlU0120.70214WQ00

[0104] In some embodiments, one or more capsid proteins are wild-type AAV2 capsid proteins. In some embodiments, an AAV2 capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0105] Delivery of codon-optimized nucleic acid sequences encoding sFasL is not limited to MCI capsid proteins. In some embodiments, an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAV9, and AAV10. In some embodiments, an AAV capsid protein is of a serotype derived from a non-human primate, for example AAVrh8 serotype. In some embodiments, the AAV capsid protein is of a serotype that has tropism for the eye of a subject, for example an AAV (e.g., AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.39 and AAVrh.43) that transduces ocular cells of a subject more efficiently than other vectors.

[0106] The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0107] In some embodiments, the instant disclosure relates to a host cell containing a nucleic acid that comprises a coding sequence encoding a protein (e.g., a sFasL protein or fragment thereof). In some embodiments, the instant disclosure relates to a composition comprising the

[0108] - 17 - #14792861vlU0120.70214WQ00

[0109] host cell described above. In some embodiments, the composition comprising the host cell above further comprises a cryopreservative.

[0110] The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No.

[0111] 5,478,745.

[0112] In some embodiments, recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with an recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the "AAV helper function" sequences (z.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild- type AAV virions (z.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein. The accessory function vector encodes nucleotide sequences for non- AAV derived viral and / or cellular functions upon which AAV is dependent for replication (z.e., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be

[0113] - 18 - #14792861vlU0120.70214WQ00

[0114] derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.

[0115] In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

[0116] A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

[0117] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0118] As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

[0119] - 19 - #14792861vlAs used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene. The foregoing methods for packaging recombinant vectors in desired AAV capsids to produce the rAAVs of the disclosure are not meant to be limiting and other suitable methods will be apparent to the skilled artisan.

[0120] rAAV-mediated delivery ofsFasL transgenes to the eye

[0121] Methods for delivering a transgene to ocular (e.g., photoreceptors, such as rod cells or cone cells, retinal cells, etc.) tissue in a subject are provided herein. The methods typically involve administering to a subject an effective amount of a rAAV comprising a nucleic acid for expressing a transgene (e.g., a sFasL protein or fragment thereof) in the subject. An “effective amount” of a rAAV is an amount sufficient to infect a sufficient number of cells of a target tissue in a subject. In some embodiments, a target tissue is ocular (e.g., photoreceptor, retinal, etc.) tissue. An effective amount of a rAAV may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to improve in the subject one or more symptoms of disease, e.g., a symptom of glaucoma. Examples of a symptom of glaucoma includes, but is not limited to, blind spots in peripheral and / or central vision, tunnel

[0122] - 20 - #14792861vlU0120.70214WQ00

[0123] vision, severe headache, eye pain, nausea and vomiting, blurred vision, halos around lights, and eye redness. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the ocular tissue to be targeted, and may thus vary among subject and tissue.

[0124] An effective amount may also depend on the rAAV used. The invention is based, in part on the recognition that certain rAAVs comprising capsid proteins mediate efficient transduction of ocular (e.g., photoreceptor, retinal, etc.) cells. In some embodiments, an rAAV used in methods provided herein comprises a capsid protein of an AAV serotype selected from the group consisting of: AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.39, and AAVrh.43. In some embodiments, an rAAV used in methods provided herein comprises an MCI capsid protein. In some embodiments, the rAAV comprises a capsid protein of AAV2 serotype (SEQ ID NO: 22). In some embodiments, the rAAV comprises a capsid protein of AAV2.MC1 serotype (SEQ ID NO: 23). In some embodiments, the capsid protein comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95% , or at least 99% identical to SEQ ID NO: 22. In some embodiments, the capsid protein comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95% , or at least 99% identical to SEQ ID NO: 23.

[0125] In certain embodiments, the effective amount of rAAV is 1010, 1011, 1012, 1013, or 1014genome copies per kg. In certain embodiments, the effective amount of rAAV is 1010, 1011, 1012, 1013, 1014, or 1015genome copies per subject.

[0126] An effective amount may also depend on the mode of administration. For example, targeting an ocular (e.g., photoreceptor, retinal, etc.) tissue by intravitreal administration, intrastromal administration, or subcutaneous injection may require different (e.g., higher or lower) doses, in some cases, than targeting an ocular (e.g., photoreceptor, retinal, etc.) tissue by another method (e.g., systemic administration, topical administration). In some embodiments, intravitreal or intrastromal injection (IS) of rAAV having certain serotypes (e.g., AAV2, AAV. MCI, etc.) mediates efficient transduction of ocular (e.g., comeal, photoreceptor, retinal, etc.) cells. In some embodiments, the injection is intrastromal injection (IS). In some embodiments, the administration is via injection, optionally subretinal injection

[0127] - 21 - #14792861vlU0120.70214WQ00

[0128] or intravitreal injection. In some embodiments, the injection is topical administration (e.g., topical administration to an eye). In some cases, multiple doses of a rAAV are administered.

[0129] In some embodiments, efficient transduction of ocular e.g., photoreceptor, retinal, etc.) cells by rAAV described herein may be useful for the treatment of a subject having glaucoma (e.g., open-angle glaucoma). Accordingly, methods and compositions for treating glaucoma are also provided herein. In some aspects, the disclosure provides a method for treating glaucoma (e.g., open-angle glaucoma), the method comprising: administering to a subject having or suspected of having glaucoma an effective amount of rAAV, wherein the rAAV comprises (i) a capsid protein having an AAV2 or MCI (e.g., AAV2.MC1) serotype, and (ii) a nucleic acid comprising a promoter operably linked to a transgene (e.g., a transgene comprising a codon-optimized sequence encoding a sFasL protein or fragment thereof as described by the disclosure).

[0130] In some embodiments, administration of a rAAV (or isolated nucleic acid) as described by the disclosure results in transduction of a retinal neuron. Examples of retinal neurons include, but are not limited to, bipolar cells, ganglion cells, horizontal cells, retina amacrine cells, rod cells and cone cells.

[0131] In some embodiments, administration of a rAAV (or isolated nucleic acid) as described by the disclosure results in transduction of a retinal ganglion cell (RGC). Examples of RGCs include, but are not limited to, W-ganglion cells, X-ganglion cells, Y-ganglion cells, midget cells, parasol cells, bistratified cells, photsensitive ganglion cells, and other ganglion cells projecting to the superior colliculus for eye movements.

[0132] Retinal ganglion cells vary significantly in terms of their size, connections, and responses to visual stimulation but they all share the defining property of having a long axon that extends into the brain. These axons form the optic nerve, optic chiasm, and optic tract. In some embodiments, administration of a rAAV (or isolated nucleic acid) as described by the disclosure prevents death of a RGC, an axon, or a combination thereof.

[0133] Methods for treating glaucoma

[0134] Methods for delivering a transgene (e.g., a gene encoding a codon-optimized sFasL protein or a fragment thereof) to a subject are provided by the disclosure. The methods typically involve administering to a subject an effective amount of a codon-optimized

[0135] - 22 - #14792861vlU0120.70214WQ00

[0136] isolated nucleic acid encoding a sFasL protein fragment, or a rAAV comprising a codon-optimized nucleic acid for expressing a sFasL protein fragment.

[0137] Aspects of the instant disclosure are based, in part, on the surprising discovery that sFasL prevents loss of RGCs and axons when expressed in a subject in need thereof, for example via administration of a viral vector (e.g., rAAV) having a capsid protein that is tropic for ocular cells, for example retinal cells, photoreceptors, etc. (e.g., MCI capsid proteins), and comprising a codon-optimized nucleic acid sequence encoding a sFasL protein fragment.

[0138] Accordingly in some aspects, the disclosure provides a transgene encoding a codon-optimized sFasL protein or a fragment thereof. A “sFasL protein fragment” refers to a functional 2 to 154 (e.g., any integer between 2 and 154) amino acid portion of a sFasL protein. In some embodiments, the sFasL protein fragment comprises a contiguous amino acid portion (e.g., amino acids 1 to 154) of sFasL (e.g., SEQ ID NO: 2). In some embodiments, the sFasL protein fragment comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) interrupted amino acid portions (e.g., amino acids 1 to 10, 32 to 120 and 64 to 150) of sFasL (e.g., SEQ ID NO: 1, 2, or 3).

[0139] In some embodiments, the codon-optimized nucleic acid sequences encoding a sFasL or fragment thereof described by the disclosure prevents loss of RGCs and axons, and are therefore useful for treating glaucoma. Generally, “glaucoma” refers to a group of eye conditions that damage the optic nerve. In some embodiments, damage to the optic nerve in glaucoma is caused by elevated pressure in the eye. Examples of glaucoma include, but are not limited to, open-angle glaucoma, angle-closure glaucoma, normal-tension glaucoma (NTG), congenital glaucoma, secondary glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, irido corneal endothelial syndrome (ICE), and uveitic glaucoma.

[0140] In some aspects, the disclosure provides a method for treating glaucoma in a subject in need thereof, the method comprising administering to a subject having glaucoma a therapeutically effective amount of an isolated nucleic acid , or a rAAV, as described by the disclosure.

[0141] An “effective amount” of a substance is an amount sufficient to produce a desired effect. In some embodiments, an effective amount of an isolated nucleic acid (e.g., an isolated

[0142] - 23 - #14792861vlU0120.70214WQ00

[0143] nucleic acid comprising a codon-optimized nucleic acid sequence encoding a sFasL protein or fragment thereof as described herein) is an amount sufficient to transfect (or infect in the context of rAAV mediated delivery) a sufficient number of target cells of a target tissue of a subject. In some embodiments, a target tissue is ocular tissue (e.g., photoreceptor cells, rod cells, cone cells, retinal ganglion cells, retinal cells, etc.). In some embodiments, an effective amount of an isolated nucleic acid (e.g., which may be delivered via an rAAV) may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to increase or supplement the expression of a gene or protein of interest (e.g., sFasL), or to improve in the subject one or more symptoms of disease (e.g., a symptom of glaucoma, such as RGC damage), etc. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue as described elsewhere in the disclosure.

[0144] As used herein, the term “treating” refers to the application or administration of a composition comprising sFasL or a fragment thereof to a subject, who has glaucoma, a symptom of glaucoma, or a predisposition toward glaucoma, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward glaucoma.

[0145] Alleviating glaucoma includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease (such as glaucoma) means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0146] "Development" or "progression" of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development

[0147] - 24 - #14792861vlU0120.70214WQ00

[0148] also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a glaucoma includes initial onset and / or recurrence.

[0149] In some embodiments, sFasL or a fragment thereof is administered to a subject in need of the treatment at an amount sufficient for lowering intraocular pressure in the subject by at least 5% (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater). In some embodiments, sFasL or a fragment thereof is administered to a subject in need of the treatment at an amount sufficient for inactivating retinal glial cells in the subject by at least 5% (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater). In some embodiments, sFasL or a fragment thereof is administered to a subject in need of the treatment at an amount sufficient for inhibiting TN Fa activity in the subject by at least 5% (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater). In some embodiments, sFasL or a fragment thereof is administered to a subject in need of the treatment at an amount sufficient for reducing retinal ganglion cell (RGC) death and / or reducing axonal degeneration in the subject by at least 5% (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater).

[0150] Methods and compositions for treating Fas ligand-dependent inflammatory conditions are also provided herein. As used herein, a “Fas ligand-dependent inflammatory condition” is a condition or a disease mediated by Fas ligand-dependent inflammation. Fas liganddependent inflammation may may be involved in ocular inflammatory diseases. Non-limiting examples of ocular inflammatory diseases include, but are not limited to, allergic conjunctivitis, uveitis, scleritis, episcleritis, optic neuritis, keratitis, orbital pseudotumor, retinal vasculitis, chronic conjunctivitis, and autoimmunity induced chronic inflammation (e.g., rheumatoid arthritis, systemic lupus erythematosus). Often, Fas ligand-dependent inflammatory conditions are linked to inflammation of the eye caused by mFasL. Without wishing to be bound by any particular theory, rAAV-based delivery of sFasL reduces inflammation of the eye in subjects having a Fas ligand-dependent inflammatory condition.

[0151] In some embodiments, the method for treating a Fas ligand-dependent inflammatory condition comprises administering to a subject in need thereof an effective amount of

[0152] - 25 - #14792861vlrecombinant adeno-associated virus (rAAV), wherein the rAAV comprises (i) capsid protein and (ii) a codon-optimized nucleic acid engineered to express sFasL or a fragment thereof.

[0153] In some embodiments, the method for treating a Fas ligand-dependent inflammatory condition, comprises detecting presence or absence of membrane-bound Fas ligand (mFasL) and / or soluble Fas ligand (sFasL) in a tissue of a subject, and treating the subject based on presence or absence of mFasL and / or sFasL, wherein treating the subject comprises administering to a subject in need thereof an effective amount of recombinant adeno-associated virus (rAAV), wherein the rAAV comprises (i) capsid protein and (ii) a codon-optimized nucleic acid engineered to express sFasL or a fragment thereof.

[0154] The skilled artisan recognizes that FasL proteins (e.g., FasL, mFasL, or sFasL) may be detected by any method known in the art. In some embodiments FasL proteins are detecting using electrophoresis, Western blot, and mass spectrometry. In some embodiments, FasL proteins are detected in ocular fluids (e.g., intraocular fluid, aqueous humor, or tears).

[0155] Combination therapy

[0156] Also provided herein are combined therapies using sFasL or a fragment thereof described herein and another anti-glaucoma therapeutic agent, such as those described herein. The term combination therapy, as used herein, embraces administration of these agents (e.g., sFasL or a fragment thereof and an anti-glaucoma therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner.

[0157] Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route including, but not limited to, rAAV-mediated delivery, oral routes, intravenous routes, intramuscular, subcutaneous routes, and direct absorption through mucous membrane tissues. The agents can be administered by the same route or by different routes. For example, a first agent (e.g., sFasL or a fragment thereof) can be administered via rAAV-mediated delivery, and a second agent (e.g., an anti-glaucoma agent) can be administered orally.

[0158] As used herein, the term “sequential” means, unless otherwise specified, characterized by a regular sequence or order, e.g., if a dosage regimen includes the administration of sFasL

[0159] - 26 - #14792861vlU0120.70214WQ00

[0160] or a fragment thereof and an anti-glaucoma agent, a sequential dosage regimen could include administration of sFasL or a fragment thereof before, simultaneously, substantially simultaneously, or after administration of the anti-glaucoma agent, but both agents will be administered in a regular sequence or order. The term “separate” means, unless otherwise specified, to keep apart one from the other. The term “simultaneously” means, unless otherwise specified, happening or done at the same time, i.e., the agents of the invention are administered at the same time. The term “substantially simultaneously” means that the agents are administered within minutes of each other (e.g., within 10 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but if the administration is consecutive it is separated in time for only a short period (e.g., the time it would take a medical practitioner to administer two agents separately). As used herein, concurrent administration and substantially simultaneous administration are used interchangeably. Sequential administration refers to temporally separated administration of the agents described herein.

[0161] Combination therapy can also embrace the administration of the agents described herein (e.g., sFasL or a fragment thereof and an anti-glaucoma agent) in further combination with other biologically active ingredients (e.g., a different anti-glaucoma agent) and non-drug therapies (e.g., surgery).

[0162] It should be appreciated that any combination of sFasL or a fragment thereof and another anti-glaucoma agent (e.g., delivered via eyedrops) may be used in any sequence for treating a glaucoma. The combinations described herein may be selected on the basis of a number of factors, which include but are not limited to the effectiveness of reducing glaucoma disease progression, lowering intraocular pressure (IOP), inactivating retinal glial cells, inhibiting TNFoc activity, reducing retinal ganglion cell (RGC) death, reducing axonal degeneration, and / or alleviating at least one symptom associated with the glaucoma, or the effectiveness for mitigating the side effects of another agent of the combination. For example, a combined therapy described herein may reduce any of the side effects associated with each individual members of the combination, for example, a side effect associated with the antiglaucoma agent.

[0163] In some embodiments, another anti-glaucoma therapeutic agent is eyedrops, an oral medication, and / or a surgical therapy. Eyedrops may comprise one or more therapeutic - 27 - #14792861vlagents, for example, prostaglandins, beta blockers, alpha- adrenergic agonists, carbonic anhydrase inhibitors, miotic agents, and cholinergic agents. In some embodiments, the oral medication comprises a carbonic anhydrase inhibitor. Examples of a surgical therapy include, but are not limited to, laser therapy, filtering surgery, drainage tubes, and electrocautery.

[0164] Pharmaceutical Compositions

[0165] The isolated nucleic acids and rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. The rAAV, preferably suspended in a physiologically compatible carrier (z.e., in a composition), may be administered to a subject, i.e. host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some embodiments, a host animal does not include a human.

[0166] Delivery of the rAAVs to a mammalian subject may be by, for example, intraocular injection or topical administration e.g., eye drops). In some embodiments, the intraocular injection is intrastromal injection, subconjunctival injection, or intravitreal injection. In some embodiments, the injection is not topical administration. Combinations of administration methods (e.g., topical administration and intrastromal injection) can also be used.

[0167] The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.

[0168] In some embodiments, a composition further comprises a pharmaceutically acceptable carrier. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

[0169] Optionally, the compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic

[0170] - 28 - #14792861vlU0120.70214WQ00

[0171] acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0172] The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue (e.g., ocular tissue, such as photoreceptor, retinal, etc., tissue) and to provide sufficient levels of gene transfer and expression without undue adverse effects. Examples of pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., subretinal delivery to the eye), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.

[0173] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors.

[0174] An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109to 1016genome copies. In some cases, a dosage between about 1011to 1013rAAV genome copies is appropriate. In certain embodiments, 109rAAV genome copies is effective to target ocular tissue e.g., comeal tissue). In some embodiments, a dose more concentrated than 109rAAV genome copies is toxic when administered to the eye of a subject. In some embodiments, an effective amount is produced by multiple doses of an rAAV.

[0175] In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar

[0176] - 29 - #14792861vlU0120.70214WQ00

[0177] week (e.g., 7 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than bi-weekly e.g., once in a two calendar week period). In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once per six calendar months. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year). In some embodiments, a dose of rAAV is administered to a subject no more than once per two calendar years (e.g., 730 days or 731 days in a leap year). In some embodiments, a dose of rAAV is administered to a subject no more than once per three calendar years (e.g., 1095 days or 1096 days in a leap year).

[0178] In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ~1013GC / ml or more). Appropriate methods for reducing aggregation of may be used, including, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)

[0179] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0180] In some embodiments, rAAVs in suitably formulated pharmaceutical compositions disclosed herein are delivered directly to target tissue, e.g., direct to ocular tissue (e.g.,

[0181] - 30 - #14792861vlU0120.70214WQ00

[0182] photoreceptor, retinal, etc., tissue). However, in certain circumstances it may be desirable to separately or in addition deliver the rAAV-based therapeutic constructs via another route, e.g., subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158;

[0183] 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver rAAVs. In some embodiments, a preferred mode of administration is by intravitreal injection or subretinal injection.

[0184] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0185] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a suitable sterile aqueous medium may be employed. For example, one dosage may be dissolved in 1

[0186] - 31 - #14792861vlU0120.70214WQ00

[0187] ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0188] Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0189] The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0190] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase

[0191] - 32 - #14792861vlU0120.70214WQ00

[0192] "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0193] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0194] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

[0195] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

[0196] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.

[0197] Alternatively, nanocapsule formulations of the rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0198] - 33 - #14792861vlU0120.70214WQ00

[0199] Exemplary embodiments of the invention will be described in more detail by the following examples. These embodiments are exemplary of the invention, which one skilled in the art will recognize is not limited to the exemplary embodiments.

[0200] EXAMPLES

[0201] Example 1: Delivery of codon-optimized sFasL

[0202] Glaucoma is the leading cause of irreversible blindness worldwide. Currently, glaucoma impacts about 80 million people worldwide, and this figure is expected to increase to 111 million people by 2040. There is currently no cure for glaucoma, and intraocular pressure (IOP) reduction is the only treatment strategy. However, lowering IOP alone does not prevent disease progression and glaucoma does not always depend on IOP. In addition, medication compliance among glaucoma patients is poor (<35%). Accordingly, there is a need for lOP-independent therapies.

[0203] This example describes codon-optimization of a natural inhibitor of ocular inflammation, soluble Fas Ligand (sFasL). FasL (CD95L) is a member of the tumor necrosis factor (TNF) family and a type II transmembrane protein. FasL signaling induces inflammation and / or apoptosis in target cells expressing the Fas receptor (CD95R), a very dangerous molecule. FasL exists in two forms: (1) membrane-bound FasL (mFasL) is proinflammatory / proapoptotic and promotes retinal ganglion cell (RGC) death; and (2) soluble FasL (sFasL) opposes the activity of mFasL (see FIG. 1). In some embodiments, codon-optimization reduces toxicity and / or improves in vivo expression of sFasL in target cells and tissues (e.g., ocular tissues). Examples of codon-optimized sFasL nucleotide sequences are shown in the section entitled REPRESENTATIVE SEQUENCES.

[0204] FIGs. 2A-2C are images that correspond to a demonstration of sFasL inhibitory activity. FIG. 2A depicts a schematic diagram of a plurality of FasL types. FIG. 2B is a plot depicting cytotoxicity of the different FasL types of FIG. 2A. FIG. 2C depicts a plurality of images indicating inflammation associated with the different FasL types of FIG. 2A.

[0205] FasL is constitutively expressed in the healthy eye. Fas is expressed on the surface of many ocular cell types, e.g., RGCs, astrocytes, microglia, muller glia, retinal pigment epithelium (RPE), and corneal endothelium and epithelium. In a healthy eye, FasL is

[0206] - 34 - #14792861vlU0120.70214WQ00

[0207] constitutively cleaved to release the neuroprotective sFasL fragment to avoid excessive cell death and inflammation. By contrast, in ocular disease, mFasL is no longer cleaved and the increased expression of this pro-apoptotic / proinflammatory isoform promotes the pathology of diseases such as glaucoma.

[0208] FIGs. 3A-3B depict a transition from sFasL to mFasL in glaucoma in a microbead-induced model of glaucoma (B6 WT mice). FIG. 3 A depicts fluorescence intensity of mFasL, sFasL, and actin 0 days and 21 days post microbead. FIG. 3B is a plot of FasL / actin ratio vs. days post microbead for sFasL and mFasL.

[0209] Regarding Fas Ligand and glaucoma: deletion of either Fas or FasL prevented axon degeneration and death of RGCs in mouse models of glaucoma; expression limited to mFasL (no sFasL) accelerated axon degeneration and death of RGCs (3 independent models of glaucoma); and AAV2. sFasL gene therapy encoding codon-optimized sFasL provided complete and sustained neuroprotection in chronic and inducible mouse models of glaucoma, even in the presence of elevated intraocular pressure. Pre-treatment with AAV2. sFasL gene therapy prevented glaucoma in chronic (FIGs. 4A-4C) and inducible (FIGs. 5A-5C) mouse models of glaucoma.

[0210] FIG. 4A depicts a DBA / 2J - chronic mouse model. At 2 months of age, an intravitreal injection AAV2. sFasL (codon-optimized) or AAV2.eGFP (3 x 109vg / 1 pl inj.) was conducted, and at 10 months of age, an RGC / axon analysis was conducted. FIG. 4B is a plot of IOP vs. age for the chronic mouse model. FIG. 4C is a plot of RGC cell density / mm2retina vs. group, wherein the groups are as follows: D2-Gp: no glaucoma; D2: Untreated DBA / 2J mice; D2-eGFP: DBA / 2J mice + AAV2-eGFP; and D2-sFasL: DBA / 2J mice + AAV2-sFasL.

[0211] Data indicate that treatment after disease onset halted disease progression. FIG. 5A is a plot for a DBA / 2J chronic mouse model. At 7 months of age, an intravitreal injection (3 x 109PFU / 1 pl inj.) was conducted, and at 15 months of age, RGC / axon analysis was conducted. FIG. 5B illustrates two plots: (1) RGC cell density / mm2vs. experimental group, and (2) axon density ( 104) / mm2vs. experimental group. The experimental groups are as follows: D2-Gp (DBA / 2J mice with single mutation, no glaucoma); D2: Untreated DBA / 2J mice; D2-eGFP: DBA / 2J mice + AAV2-eGFP; and D2-sFasL: DBA / 2J mice treated with AAV2-sFasL.

[0212] - 35 - #14792861vlU0120.70214WQ00

[0213] Treatment post injury restored visual activity. FIG. 6A is a plot of IOP vs. days post microbead injection for a microbead - inducible model. FIG. 6B is a plot of amplitude vs. condition (four conditions), as an electrophysiological assessment of RGC function.

[0214] The approach of the inventors in this study is set apart from others. A single intravitreal injection of codon-optimized AAV2-sFasL provided long-term protection, even in the presence of elevated IOP. Because the sFasL utilized in this example is released extracellularly, not every RGC needed to be transduced in order to achieve protection.

[0215] Multiple pathways were targeted, including activation of glial cells, inflammation, and apoptosis. The potential for manipulation of FasL isoforms was not restricted to glaucoma; experimental data supported a broad set of applications throughout the eye, including age-related macular degeneration, corneal keratitis, comeal transplantation, and RGC transplantation.

[0216] Example 2: Preclinical model of canine glaucoma

[0217] A genetically programmed model of canine primary open-angle glaucoma has been developed. Glaucoma is a common disease in dogs. A dog eye has similarities to human eye, e.g., inner limiting membrane, and comparable inflammatory response to AAV. Reagents utilized include canine sFasL expression vector and anti-canine FasL antisera. An improved AAV vector was developed, having a novel capsid variant, MCI capsid, and codon optimization and CpG reduction.

[0218] Results for AAV2-MCl-CB6-PI-eGFP include FIG. 7A, a fundic image revealing that tagging was strongest in the superior nasal region. FIG. 7B is a light microscope image of a cross-section of FIG. 7A indicating high expression throughout the retinal layers from an outer nerve fiber layer to a retinal pigment epithelium layer.

[0219] Results for AAV2-7m8-CB6-PI-eGFP include FIG. 8A, a fundic image from 6 weeks post-injection revealing very strong expression in a superior nasal region. FIG. 8B is a light microscope image of a cross-section of FIG. 8A indicating inner nuclear layer tagging, nerve fiber layer, and inner plexiform layer.

[0220] Results for AAV2-CB6-eGFP include FIG. 9 A, a fundic image showing GFP expression around the optic nerve head. FIG. 9B is a light microscope image of a crosssection of FIG. 9 A showing little expression and one spot of tagging in inner nuclear layer.

[0221] - 36 - #14792861vlFIG. 10 shows representative data indicating treatment of subjects with AAV-sFasL after disease onset halts disease progression.

[0222] FIG. 11 shows representative data from an AAV2-sFasL Pilot dog study. Briefly, delivery of 1011vg / eye led to good expression but was associated with uveitis, retinitis, and optic neuritis. Lowering the AAV-sFasL dose to IO10and 109vg / eye reduced inflammation but also reduced sFasL expression and was not neuroprotective.

[0223] FIG. 12 shows representative data indicating MCI AAV2 capsid protein mediates improved transduction and reduced immunogenicity when compared to AAV2 and AAV2.7m8 capsid-containing AAVs.

[0224] Example 3: Comparison of AAV2 mouse FasL to AAV canine FasL

[0225] Treatment with an AAV2 mouse FasL was compared to an AAV canine FasL in a microbead (MB) murine model of glaucoma. Briefly, mice were injected with control and MB mice were administered (e.g., by intravitreal injection) either empty rAAV vector, AAV-mouse sFasL, or AAV-canine sFasL. FIG. 13A shows, e.g., that intraocular pressure increased in all the MB-injected mice. FIG. 13B shows loss of ganglion cells, which are a readout for glaucoma in empty vector-treated mice, but not in the mouse sFasL-treated mice or the canine sFasL-treated mice. This data indicates that the AAV-delivered canine sFasL binds mouse Fas and that canine sFasL functions in animal models of glaucoma. It was also observed that AAV2 effectively delivers sFasL in mice.

[0226] REPRESENTATIVE SEQUENCES

[0227] >SEQ ID NO: 1 - Canine Soluble Fas Ligand (Accession No.: AAT37155.1) Protein Sequence GKPNSRSIPLEWEDTYGIALVSGVKYKKGGLVINDTGLYFVYSKVYFRGQSCNNKPL NHKVYMRNSKYPQDLMLMEGKIMNYCTTGQMWARSSYLGAVFNLTSADHLYVNV SELSLVSFEESKTFFGLYKL

[0228] >SEQ ID NO: 2 - Human Soluble Fas Ligand (Accession No.: P48023.1; Amino Acids 128-281) Protein Sequence EKQIGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINE TGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMW ARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL

[0229] - 37 - #14792861vlU0120.70214WQ00

[0230] >SEQ ID NO: 3 - Mouse Soluble Fas Ligand (Accession Number AAB02915.1; Amino acids 126-279) Protein Sequence EKQIANPSTPSEKKEPRSVAHLTGNPHSRSIPLEWEDTYGTALISGVKYKKGGLVINEA GLYFVYSKVYFRGQSCNNQPLNHKVYMRNSKYPGDLVLMEEKRLNYCTTGQIWAH SSYLGAVFNLTSADHLYVNISQLSLINFEESKTFFGLYKL

[0231] >SEQ ID NO: 4 - Codon-optimized canine sFasL nucleic acid sequence 1 CTTAAGCTTGCCGCCACCATGCGGGCTTGGATTTTCTTCCTGCTGTGTCTGGCTGG CAGAGCCCTGGCCCTCGAGAAGCAGATCGGCCAGCCTAATCCTCCAAGCGAGAA AAGAGAGCTGAGAAAGGTGGCCCACCTGACCGGAAAGCCCAATAGCCGGTCCAT CCCTCTGGAATGGGAGGACACCTACGGCATCGCCCTGGTGTCTGGTGTTAAGTAC AAGAAAGGAGGCCTGGTCATCAACGACACAGGCCTGTACTTCGTGTACAGCAAA GTGTACTTTCGGGGCCAAAGCTGCAACAACAAGCCTCTGAACCACAAGGTGTATA TGAGAAACAGCAAGTACCCCCAGGACCTGATGCTGATGGAAGGCAAGATCATGA ACTACTGCACCACCGGCCAGATGTGGGCCAGAAGCAGCTACCTGGGCGCCGTGTT CAACCTGACCAGCGCCGATCACCTGTACGTGAACGTGTCTGAGCTGTCTCTGGTG TCCTTCGAGGAAAGCAAGACATTTTTCGGCCTGTATAAGCTGTGATCTAGAGGGCC

[0232] C

[0233] >SEQ ID NO: 5 - Codon-optimized canine sFasL nucleic acid sequence 2 (CpG Null) CTTAAGCTTGCCGCCACCATGAGGGCTTGGATTTTCTTCCTGCTGTGTCTGGCTGG CAGAGCCCTGGCCCTGGAGAAGCAGATTGGCCAGCCTAATCCTCCAAGTGAGAA AAGAGAGCTGAGAAAGGTGGCCCACCTGACAGGAAAGCCCAATAGCAGGTCCAT CCCTCTGGAATGGGAGGACACCTATGGCATTGCCCTGGTGTCTGGTGTTAAGTACA AGAAAGGAGGCCTGGTCATCAATGACACAGGCCTGTACTTTGTGTACAGCAAAGT GTACTTTAGGGGCCAAAGCTGCAACAACAAGCCTCTGAACCACAAGGTGTATATG AGAAACAGCAAGTACCCCCAGGACCTGATGCTGATGGAAGGCAAGATCATGAACT ACTGCACCACAGGCCAGATGTGGGCCAGAAGCAGCTACCTGGGGGCTGTGTTCA ACCTGACCAGTGCTGATCACCTGTATGTGAATGTGTCTGAGCTGTCTCTGGTGTCC TTTGAGGAAAGCAAGACATTTTTTGGCCTGTATAAGCTGTGATCTAGAGGGCCC

[0234] >SEQ ID NO: 6 - Codon-optimized canine sFasL nucleic acid sequence 3 (High Priority) CTTAAGCTTGCCGCCACCATGCGGGCTTGGATTTTCTTCCTGCTGTGTCTGGCTGG CAGAGCCCTGGCCCTCGAGAAGCAGATCGGCCAGCCTAATCCTCCAAGCGAGAA AAGAGAGCTGAGAAAGGTGGCCCACCTGACCGGAAAGCCCAATAGCCGGTCCAT CCCTCTGGAATGGGAGGACACCTATGGCATCGCCCTGGTGTCTGGTGTTAAGTACA AGAAAGGAGGCCTGGTCATCAATGACACAGGCCTGTACTTTGTGTACAGCAAAGT GTACTTTCGGGGCCAAAGCTGCAACAACAAGCCTCTGAACCACAAGGTGTATATG AGAAACAGCAAGTACCCCCAGGACCTGATGCTGATGGAAGGCAAGATCATGAACT ACTGCACCACCGGCCAGATGTGGGCCAGAAGCAGCTACCTGGGCGCTGTGTTCAA CCTGACCAGCGCCGATCACCTGTATGTGAATGTGTCTGAGCTGTCTCTGGTGTCCT TCGAGGAAAGCAAGACATTTTTCGGCCTGTATAAGCTGTGATCTAGAGGGCCC

[0235] >SEQ ID NO: 7 - Codon-optimized human sFasL nucleic acid sequence 1 CTTAAGCTTGCCGCCACCATGCGGGCCTGGATCTTCTTCCTGCTGTGTCTGGCTGG CAGAGCTCTGGCCCTGGAAAAGCAGATCGGCCACCCCAGCCCACCTCCTGAGAA

[0236] - 38 - #14792861vlU0120.70214WQ00

[0237] GAAAGAGCTGAGAAAGGTGGCCCACCTGACAGGAAAAAGCAATAGCAGAAGCA TGCCTCTGGAATGGGAGGACACCTACGGCATCGTGCTGCTCTCCGGCGTGAAGTA CAAGAAGGGCGGACTTGTTATCAACGAGACAGGCCTGTACTTCGTGTACAGCAA GGTCTACTTTAGGGGCCAGAGCTGCAACAACCTGCCTCTGTCCCATAAGGTGTAC ATGCGGAACAGCAAATACCCCCAGGACCTGGTGATGATGGAAGGCAAGATGATG AGCTACTGCACCACCGGCCAAATGTGGGCCAGATCTTCTTACCTGGGAGCCGTGT TCAACCTGACCAGCGCCGATCACCTGTACGTGAATGTGTCCGAGCTGAGCCTGGT GAACTTCGAGGAAAGCCAGACCTTCTTTGGCCTGTATAAGCTGTGATCTAGAGGG CCC

[0238] >SEQ ID NO: 8 - Codon-optimized human sFasL nucleic acid sequence 2 (CpG Null) CTTAAGCTTGCCGCCACCATGAGAGCCTGGATCTTCTTCCTGCTGTGTCTGGCTGG CAGAGCTCTGGCCCTGGAAAAGCAGATTGGCCACCCCAGCCCACCTCCTGAGAA GAAAGAGCTGAGAAAGGTGGCCCACCTGACAGGAAAAAGCAATAGCAGAAGCA TGCCTCTGGAATGGGAGGACACCTATGGCATTGTGCTGCTCTCTGGAGTGAAGTA CAAGAAGGGAGGACTTGTTATCAATGAGACAGGCCTGTACTTTGTGTACAGCAA GGTCTACTTTAGGGGCCAGAGCTGCAACAACCTGCCTCTGTCCCATAAGGTGTAC ATGAGAAACAGCAAATACCCCCAGGACCTGGTGATGATGGAAGGCAAGATGATG AGCTACTGCACCACAGGCCAAATGTGGGCCAGATCTTCTTACCTGGGAGCTGTGT TCAACCTGACCAGTGCTGATCACCTGTATGTGAATGTGTCTGAGCTGAGCCTGGT GAACTTTGAGGAAAGCCAGACCTTCTTTGGCCTGTATAAGCTGTGATCTAGAGGG CCC

[0239] >SEQ ID NO: 9 - Codon-optimized human sFasL nucleic acid sequence 3 (High Priority) CTTAAGCTTGCCGCCACCATGCGGGCCTGGATCTTCTTCCTGCTGTGTCTGGCTGG CAGAGCTCTGGCCCTGGAAAAGCAGATCGGCCACCCCAGCCCACCTCCTGAGAA GAAAGAGCTGAGAAAGGTGGCCCACCTGACAGGAAAAAGCAATAGCAGAAGCA TGCCTCTGGAATGGGAGGACACCTATGGCATTGTGCTGCTCTCCGGCGTGAAGTA CAAGAAGGGCGGACTTGTTATCAATGAGACAGGCCTGTACTTTGTGTACAGCAA GGTCTACTTTAGGGGCCAGAGCTGCAACAACCTGCCTCTGTCCCATAAGGTGTAC ATGCGGAACAGCAAATACCCCCAGGACCTGGTGATGATGGAAGGCAAGATGATG AGCTACTGCACCACCGGCCAAATGTGGGCCAGATCTTCTTACCTGGGAGCTGTGT TCAACCTGACCAGCGCCGATCACCTGTATGTGAATGTGTCCGAGCTGAGCCTGGT GAACTTCGAGGAAAGCCAGACCTTCTTTGGCCTGTATAAGCTGTGATCTAGAGGG CCC

[0240] >SEQ ID NO: 10 - Codon-optimized mouse sFasL nucleic acid sequence 1 CTTAAGCTTGCCGCCACCATGAGAGCCTGGATCTTCTTCCTGCTGTGCCTGGCTG GCAGAGCCCTCGCCCTGGAAAAGCAGATCGCCAACCCTTCTACACCTAGCGAGA AGAAGGAACCAAGAAGCGTGGCCCATCTTACCGGCAACCCCCACAGCAGAAGCA TCCCTCTGGAATGGGAGGACACCTACGGCACCGCCCTGATCTCCGGCGTGAAAT ACAAGAAGGGCGGCCTGGTCATCAACGAGACAGGCCTGTACTTTGTGTACAGCA AAGTGTATTTCCGCGGACAAAGCTGTAACAACCAGCCTCTGAATCACAAGGTGT ACATGCGGAACAGCAAGTACCCCGAGGATCTGGTGCTGATGGAAGAGAAGCGGC TGAACTACTGCACCACCGGCCAGATTTGGGCTCACAGCAGCTACCTGGGAGCCG TGTTTAACCTGACAAGCGCCGACCACCTGTACGTGAACATCTCTCAGCTGAGCCT

[0241] - 39 - #14792861vlU0120.70214WQ00

[0242] GATCAATTTCGAGGAGAGCAAGACCTTCTTCGGCCTGTACAAACTGTGATCTAGA GGGCCC

[0243] >SEQ ID NO: 11 - Codon-optimized mouse sFasL nucleic acid sequence 2 (CpG Null) CTTAAGCTTGCCGCCACCATGAGAGCCTGGATCTTCTTCCTGCTGTGCCTGGCTG GCAGAGCCCTGGCCCTGGAAAAGCAGATTGCCAACCCTTCTACACCTAGTGAGA AGAAGGAACCAAGAAGTGTGGCCCATCTTACAGGCAACCCCCACAGCAGAAGCA TCCCTCTGGAATGGGAGGACACCTATGGCACAGCCCTGATCTCTGGAGTGAAATA CAAGAAGGGAGGCCTGGTCATCAATGAGACAGGCCTGTACTTTGTGTACAGCAA AGTGTATTTCAGAGGACAAAGCTGTAACAACCAGCCTCTGAATCACAAGGTGTA CATGAGGAACAGCAAGTACCCTGAGGATCTGGTGCTGATGGAAGAGAAGAGGCT GAACTACTGCACCACAGGCCAGATTTGGGCTCACAGCAGCTACCTGGGAGCTGT GTTTAACCTGACATCTGCTGACCACCTGTATGTGAACATCTCTCAGCTGAGCCTG ATCAATTTTGAGGAGAGCAAGACCTTCTTTGGCCTGTACAAACTGTGATCTAGAG GGCCC

[0244] >SEQ ID NO: 12 - Codon-optimized mouse sFasL nucleic acid sequence 3 (High Priority) CTTAAGCTTGCCGCCACCATGAGAGCCTGGATCTTCTTCCTGCTGTGCCTGGCTG GCAGAGCCCTCGCCCTGGAAAAGCAGATCGCCAACCCTTCTACACCTAGCGAGA AGAAGGAACCAAGAAGCGTGGCCCATCTTACCGGCAACCCCCACAGCAGAAGCA TCCCTCTGGAATGGGAGGACACCTATGGCACCGCCCTGATCTCCGGCGTGAAATA CAAGAAGGGCGGCCTGGTCATCAATGAGACAGGCCTGTACTTTGTGTACAGCAA AGTGTATTTCCGCGGACAAAGCTGTAACAACCAGCCTCTGAATCACAAGGTGTAC ATGCGGAACAGCAAGTACCCCGAGGATCTGGTGCTGATGGAAGAGAAGCGGCTG AACTACTGCACCACCGGCCAGATTTGGGCTCACAGCAGCTACCTGGGAGCTGTGT TTAACCTGACAAGCGCCGACCACCTGTATGTGAACATCTCTCAGCTGAGCCTGAT CAATTTCGAGGAGAGCAAGACCTTCTTCGGCCTGTACAAACTGTGATCTAGAGGG CCC

[0245] >SEQ ID NO: 13 - pAAV-Canine-FasL ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcaattcacgcgtcgacattgattattgactagctctggtcgttac ataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatg ggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtg cggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaag cgcgcggcgggcgggagcgggatcagccaccgcggtggcggcctagagtcgacgaggaactgaaaaaccagaaagttaactgg taagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGCGGGCTTGGATT TTCTTCCTGCTGTGTCTGGCTGGCAGAGCCCTGGCCCTCGAGAAGCAGATCGGCC AGCCTAATCCTCCAAGCGAGAAAAGAGAGCTGAGAAAGGTGGCCCACCTGACCG GAAAGCCCAATAGCCGGTCCATCCCTCTGGAATGGGAGGACACCTACGGCATCGC CCTGGTGTCTGGTGTTAAGTACAAGAAAGGAGGCCTGGTCATCAACGACACAGGC CTGTACTTCGTGTACAGCAAAGTGTACTTTCGGGGCCAAAGCTGCAACAACAAGC CTCTGAACCACAAGGTGTATATGAGAAACAGCAAGTACCCCCAGGACCTGATGCT

[0246] - 40 - #14792861vlU0120.70214WQ00

[0247] GATGGAAGGCAAGATCATGAACTACTGCACCACCGGCCAGATGTGGGCCAGAAG CAGCTACCTGGGCGCCGTGTTCAACCTGACCAGCGCCGATCACCTGTACGTGAAC GTGTCTGAGCTGTCTCTGGTGTCCTTCGAGGAAAGCAAGACATTTTTCGGCCTGTA TAAGCTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttctagttgc cagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgc atcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcct aggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcg ctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagcctt aattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcccc ctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcg ccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcct ttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgtt ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgc cgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcact tttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgc ttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctca cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaa gatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgc cgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggacc gaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaa acgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccg gcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacga cggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagacca agtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatc ccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag gcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagc gtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt tctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcag ctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggc tttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgcca gatttaattaaggccttaattagg

[0248] >SEQ ID NO: 14 - pAAV-Canine-FasL-CpG Null ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcaattcacgcgtcgacattgattattgactagctctggtcgttac ataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac

[0249] - 41 - #14792861vlU0120.70214WQ00

[0250] gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatg ggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtg cggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaag cgcgcggcgggcgggagcgggatcagccaccgcggtggcggcctagagtcgacgaggaactgaaaaaccagaaagttaactgg taagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGAGGGCTTGGATT TTCTTCCTGCTGTGTCTGGCTGGCAGAGCCCTGGCCCTGGAGAAGCAGATTGGCC AGCCTAATCCTCCAAGTGAGAAAAGAGAGCTGAGAAAGGTGGCCCACCTGACAG GAAAGCCCAATAGCAGGTCCATCCCTCTGGAATGGGAGGACACCTATGGCATTGC CCTGGTGTCTGGTGTTAAGTACAAGAAAGGAGGCCTGGTCATCAATGACACAGGC CTGTACTTTGTGTACAGCAAAGTGTACTTTAGGGGCCAAAGCTGCAACAACAAGC CTCTGAACCACAAGGTGTATATGAGAAACAGCAAGTACCCCCAGGACCTGATGCT GATGGAAGGCAAGATCATGAACTACTGCACCACAGGCCAGATGTGGGCCAGAAG CAGCTACCTGGGGGCTGTGTTCAACCTGACCAGTGCTGATCACCTGTATGTGAATG TGTCTGAGCTGTCTCTGGTGTCCTTTGAGGAAAGCAAGACATTTTTTGGCCTGTAT AAGCTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttctagttgcc agccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgca tcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagccta ggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgc tcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagcctta attaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccc tttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgc cctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctt tcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttta cggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttg gagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgcc gatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcactt ttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgct tcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctca cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaa gatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgc cgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggacc gaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaa acgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccg gcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacga cggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagacca agtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatc ccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag gcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagc gtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat

[0251] - 42 - #14792861vlU0120.70214WQ00

[0252] gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt tctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcag ctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggc tttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgcca gatttaattaaggccttaattagg

[0253] >SEQ ID NO: 15 - pAAV-Canine-FasL-CpG High Priority (HP) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcaattcacgcgtcgacattgattattgactagctctggtcgttac ataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggt gcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaa gcgcgcggcgggcgggagcgggatcagccaccgcggtggcggcctagagtcgacgaggaactgaaaaaccagaaagttaactg gtaagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGCGGGCTTGGAT TTTCTTCCTGCTGTGTCTGGCTGGCAGAGCCCTGGCCCTCGAGAAGCAGATCGGC CAGCCTAATCCTCCAAGCGAGAAAAGAGAGCTGAGAAAGGTGGCCCACCTGACC GGAAAGCCCAATAGCCGGTCCATCCCTCTGGAATGGGAGGACACCTATGGCATCG CCCTGGTGTCTGGTGTTAAGTACAAGAAAGGAGGCCTGGTCATCAATGACACAGG CCTGTACTTTGTGTACAGCAAAGTGTACTTTCGGGGCCAAAGCTGCAACAACAAG CCTCTGAACCACAAGGTGTATATGAGAAACAGCAAGTACCCCCAGGACCTGATGC TGATGGAAGGCAAGATCATGAACTACTGCACCACCGGCCAGATGTGGGCCAGAAG CAGCTACCTGGGCGCTGTGTTCAACCTGACCAGCGCCGATCACCTGTATGTGAATG TGTCTGAGCTGTCTCTGGTGTCCTTCGAGGAAAGCAAGACATTTTTCGGCCTGTAT AAGCTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttctagttgcc agccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgca tcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagccta ggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgc tcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagcctta attaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccc tttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgc cctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctt tcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttta cggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttg gagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgcc gatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcactt ttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgct tcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctca cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaa gatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgc cgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggacc gaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaa

[0254] - 43 - #14792861vlU0120.70214WQ00

[0255] acgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccg gcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacga cggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagacca agtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatc ccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag gcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagc gtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt tctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcag ctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggc tttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgcca gatttaattaaggccttaattagg

[0256] >SEQ ID NO: 16 - pAAV-Human-FasL ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtcgacattgattattgactctggtcgtta cataacttacggtaaatggcccgcctggctgaccgcccaacgaccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgg agaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaa agcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggcggccctagagtcgatcgaggaactgaaaaaccagaa agttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGCGGGC CTGGATCTTCTTCCTGCTGTGTCTGGCTGGCAGAGCTCTGGCCCTGGAAAAGCAG ATCGGCCACCCCAGCCCACCTCCTGAGAAGAAAGAGCTGAGAAAGGTGGCCCAC CTGACAGGAAAAAGCAATAGCAGAAGCATGCCTCTGGAATGGGAGGACACCTAC GGCATCGTGCTGCTCTCCGGCGTGAAGTACAAGAAGGGCGGACTTGTTATCAAC GAGACAGGCCTGTACTTCGTGTACAGCAAGGTCTACTTTAGGGGCCAGAGCTGC AACAACCTGCCTCTGTCCCATAAGGTGTACATGCGGAACAGCAAATACCCCCAG GACCTGGTGATGATGGAAGGCAAGATGATGAGCTACTGCACCACCGGCCAAATG TGGGCCAGATCTTCTTACCTGGGAGCCGTGTTCAACCTGACCAGCGCCGATCACC TGTACGTGAATGTGTCCGAGCTGAGCCTGGTGAACTTCGAGGAAAGCCAGACCTT CTTTGGCCTGTATAAGCTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcc tcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcc taataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagg attgggaagacaattaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctc tgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagc gagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg

[0257] - 44 - #14792861vlU0120.70214WQ00

[0258] gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtt tttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgctta caatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttg ccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgga tctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtat tatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaa aagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctga atgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactac ttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatc gtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattg gtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataat ctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccg aaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaac tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggt cggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggcc gattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcatt aggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatga ccatgattacgccagatttaattaaggccttaattagg

[0259] >SEQ ID NO: 17 - pAAV-Human-FasL-CpG Null ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtcgacattgattattgactctggtcgtta cataacttacggtaaatggcccgcctggctgaccgcccaacgaccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgg agaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaa agcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggcggccctagagtcgatcgaggaactgaaaaaccagaa agttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGAGAGC CTGGATCTTCTTCCTGCTGTGTCTGGCTGGCAGAGCTCTGGCCCTGGAAAAGCAG ATTGGCCACCCCAGCCCACCTCCTGAGAAGAAAGAGCTGAGAAAGGTGGCCCAC CTGACAGGAAAAAGCAATAGCAGAAGCATGCCTCTGGAATGGGAGGACACCTAT GGCATTGTGCTGCTCTCTGGAGTGAAGTACAAGAAGGGAGGACTTGTTATCAATG

[0260] - 45 - #14792861vlU0120.70214WQ00

[0261] AGACAGGCCTGTACTTTGTGTACAGCAAGGTCTACTTTAGGGGCCAGAGCTGCAA CAACCTGCCTCTGTCCCATAAGGTGTACATGAGAAACAGCAAATACCCCCAGGA CCTGGTGATGATGGAAGGCAAGATGATGAGCTACTGCACCACAGGCCAAATGTG GGCCAGATCTTCTTACCTGGGAGCTGTGTTCAACCTGACCAGTGCTGATCACCTG TATGTGAATGTGTCTGAGCTGAGCCTGGTGAACTTTGAGGAAAGCCAGACCTTCT TTGGCCTGTATAAGCTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcctcg actgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaa taaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggatt gggaagacaattaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctg cgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcga gcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttg cagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcg aatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcccta gcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttc gccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgattta taagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaa tttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataa ccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcct tcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatct caacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattat cccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaa gcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaac gatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaat gaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactt actctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctg gtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgt agttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggt aactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatct catgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacc gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgata gttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactg agatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcg gaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcg tcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcc ttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccg aacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgat tcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattagg caccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacca tgattacgccagatttaattaaggccttaattagg

[0262] >SEQ ID NO: 18 - pAAV-Human-FasL-CpG HP ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtcgacattgattattgactctggtcgtta

[0263] - 46 - #14792861vlU0120.70214WQ00

[0264] cataacttacggtaaatggcccgcctggctgaccgcccaacgaccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgg agaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaa agcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggcggccctagagtcgatcgaggaactgaaaaaccagaa agttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGCGGGC CTGGATCTTCTTCCTGCTGTGTCTGGCTGGCAGAGCTCTGGCCCTGGAAAAGCAG ATCGGCCACCCCAGCCCACCTCCTGAGAAGAAAGAGCTGAGAAAGGTGGCCCAC CTGACAGGAAAAAGCAATAGCAGAAGCATGCCTCTGGAATGGGAGGACACCTAT GGCATTGTGCTGCTCTCCGGCGTGAAGTACAAGAAGGGCGGACTTGTTATCAATG AGACAGGCCTGTACTTTGTGTACAGCAAGGTCTACTTTAGGGGCCAGAGCTGCAA CAACCTGCCTCTGTCCCATAAGGTGTACATGCGGAACAGCAAATACCCCCAGGA CCTGGTGATGATGGAAGGCAAGATGATGAGCTACTGCACCACCGGCCAAATGTG GGCCAGATCTTCTTACCTGGGAGCTGTGTTCAACCTGACCAGCGCCGATCACCTG TATGTGAATGTGTCCGAGCTGAGCCTGGTGAACTTCGAGGAAAGCCAGACCTTCT TTGGCCTGTATAAGCTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcctcg actgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaa taaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggatt gggaagacaattaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctg cgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcga gcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttg cagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcg aatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcccta gcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttc gccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgattta taagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaa tttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataa ccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcct tcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatct caacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattat cccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaa gcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaac gatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaat gaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactt actctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctg gtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgt agttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggt aactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatct catgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacc gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgata gttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactg

[0265] - 47 - #14792861vlU0120.70214WQ00

[0266] agatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcg gaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcg tcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcc ttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccg aacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgat tcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattagg caccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacca tgattacgccagatttaattaaggccttaattagg

[0267] >SEQ ID NO: 19 - pAAV-Mouse-FasL ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtcgacattgattattgactctggtcgtta cataacttacggtaaatggcccgcctggctgaccgcccaacgaccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgg agaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaa agcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggcggccctagagtcgatcgaggaactgaaaaaccagaa agttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGAGAGC CTGGATCTTCTTCCTGCTGTGCCTGGCTGGCAGAGCCCTCGCCCTGGAAAAGCAG ATCGCCAACCCTTCTACACCTAGCGAGAAGAAGGAACCAAGAAGCGTGGCCCAT CTTACCGGCAACCCCCACAGCAGAAGCATCCCTCTGGAATGGGAGGACACCTAC GGCACCGCCCTGATCTCCGGCGTGAAATACAAGAAGGGCGGCCTGGTCATCAAC GAGACAGGCCTGTACTTTGTGTACAGCAAAGTGTATTTCCGCGGACAAAGCTGTA ACAACCAGCCTCTGAATCACAAGGTGTACATGCGGAACAGCAAGTACCCCGAGG ATCTGGTGCTGATGGAAGAGAAGCGGCTGAACTACTGCACCACCGGCCAGATTT GGGCTCACAGCAGCTACCTGGGAGCCGTGTTTAACCTGACAAGCGCCGACCACC TGTACGTGAACATCTCTCAGCTGAGCCTGATCAATTTCGAGGAGAGCAAGACCTT CTTCGGCCTGTACAAACTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcc tcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcc taataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagg attgggaagacaattaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctc tgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagc gagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtt tttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgctta caatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttg ccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgga tctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtat tatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaa

[0268] - 48 - #14792861vlU0120.70214WQ00

[0269] aagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctga atgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactac ttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatc gtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattg gtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataat ctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccg aaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaac tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggt cggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggcc gattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcatt aggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatga ccatgattacgccagatttaattaaggccttaattagg

[0270] >SEQ ID NO: 20 - pAAV-Mouse-FasL-CpG Null ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtcgacattgattattgactctggtcgtta cataacttacggtaaatggcccgcctggctgaccgcccaacgaccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgg agaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaa agcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggcggccctagagtcgatcgaggaactgaaaaaccagaa agttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGAGAGC CTGGATCTTCTTCCTGCTGTGCCTGGCTGGCAGAGCCCTGGCCCTGGAAAAGCAG ATTGCCAACCCTTCTACACCTAGTGAGAAGAAGGAACCAAGAAGTGTGGCCCAT CTTACAGGCAACCCCCACAGCAGAAGCATCCCTCTGGAATGGGAGGACACCTAT GGCACAGCCCTGATCTCTGGAGTGAAATACAAGAAGGGAGGCCTGGTCATCAAT GAGACAGGCCTGTACTTTGTGTACAGCAAAGTGTATTTCAGAGGACAAAGCTGT AACAACCAGCCTCTGAATCACAAGGTGTACATGAGGAACAGCAAGTACCCTGAG GATCTGGTGCTGATGGAAGAGAAGAGGCTGAACTACTGCACCACAGGCCAGATT TGGGCTCACAGCAGCTACCTGGGAGCTGTGTTTAACCTGACATCTGCTGACCACC TGTATGTGAACATCTCTCAGCTGAGCCTGATCAATTTTGAGGAGAGCAAGACCTT CTTTGGCCTGTACAAACTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcc tcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcc taataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagg attgggaagacaattaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctc tgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagc

[0271] - 49 - #14792861vlU0120.70214WQ00

[0272] gagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtt tttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgctta caatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttg ccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgga tctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtat tatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaa aagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctga atgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactac ttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatc gtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattg gtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataat ctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccg aaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaac tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggt cggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggcc gattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcatt aggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatga ccatgattacgccagatttaattaaggccttaattagg

[0273] >SEQ ID NO: 21 - pAAV-Mouse-FasL-CpG HP ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtcgacattgattattgactctggtcgtta cataacttacggtaaatggcccgcctggctgaccgcccaacgaccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgg agaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaa agcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggcggccctagagtcgatcgaggaactgaaaaaccagaa agttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccCTTAAGCTTGCCGCCACCATGAGAGC CTGGATCTTCTTCCTGCTGTGCCTGGCTGGCAGAGCCCTCGCCCTGGAAAAGCAG ATCGCCAACCCTTCTACACCTAGCGAGAAGAAGGAACCAAGAAGCGTGGCCCAT

[0274] - 50 - #14792861vlU0120.70214WQ00

[0275] CTTACCGGCAACCCCCACAGCAGAAGCATCCCTCTGGAATGGGAGGACACCTAT GGCACCGCCCTGATCTCCGGCGTGAAATACAAGAAGGGCGGCCTGGTCATCAAT GAGACAGGCCTGTACTTTGTGTACAGCAAAGTGTATTTCCGCGGACAAAGCTGTA ACAACCAGCCTCTGAATCACAAGGTGTACATGCGGAACAGCAAGTACCCCGAGG ATCTGGTGCTGATGGAAGAGAAGCGGCTGAACTACTGCACCACCGGCCAGATTT GGGCTCACAGCAGCTACCTGGGAGCTGTGTTTAACCTGACAAGCGCCGACCACC TGTATGTGAACATCTCTCAGCTGAGCCTGATCAATTTCGAGGAGAGCAAGACCTT CTTCGGCCTGTACAAACTGTGATCTAGAGGGCCCatcgataccgtcgactagagctcgctgatcagcc tcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcc taataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagg attgggaagacaattaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctc tgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagc gagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtt tttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgctta caatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttg ccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgga tctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtat tatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaa aagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctga atgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactac ttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatc gtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattg gtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataat ctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccg aaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaac tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggt cggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggcc gattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcatt aggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatga ccatgattacgccagatttaattaaggccttaattagg

[0276] >SEQ ID NO: 22 - AAV type 2 Capsid Protein Sequence

[0277] - 51 - #14792861vlU0120.70214WQ00

[0278] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPF NGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFG GNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPA RKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVG NSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIAN NLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT NTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTG ATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITD EEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPI WAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTR NL

[0279] >SEQ ID NO: 23 - AAV2-MC1 Capsid Protein Sequence- E36G, D80N, VI 25 A MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAGRHKDDSRGLVLPGYKYLGP FNGLDKGEPVNEADAAALEHDKAYNRQLDSGDNPYLKYNHADAEFQERLKEDTSF GGNLGRAVFQAKKRALEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQP ARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGV GNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYST PWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIA NNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGR SSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSR TNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMIT DEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQG PIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYST GQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLT RNL

[0280] OTHER EMBODIMENTS

[0281] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the present disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0282] - 52 - #14792861vlU0120.70214WQ00

[0283] EQUIVALENTS AND SCOPE

[0284] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0285] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0286] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of

[0287] - 53 - #14792861vlthe present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0288] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0289] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

[0290] - 54 - #14792861vl

Claims

U0120.70214WQ00CLAIMSWhat is claimed is:

1. An isolated nucleic acid comprising a transgene flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), the transgene comprising:(i) a promoter operably linked to a nucleic acid sequence encoding a codon-optimized soluble Fas Ligand (sFasL) peptide.

2. The isolated nucleic acid of claim 1, wherein the codon-optimized sFasL peptide is a mammalian sFasL peptide.

3. The isolated nucleic acid of claim 1 or claim 2, wherein the codon-optimized sFasL peptide is a canine sFasL peptide, a human sFasL peptide, or a mouse sFasL peptide.

4. The isolated nucleic acid of any one of claims 1 to 3, wherein the sFasL peptide comprises the amino acid sequence in any one of SEQ ID NOs: 1-3.

5. The isolated nucleic acid of any one of claims 1 to 4, wherein the nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NOs: 4-6.

6. The isolated nucleic acid of any one of claims 1 to 4, wherein the nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NOs: 7-9.

7. The isolated nucleic acid of any one of claims 1 to 4, wherein the nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NOs: 10-12.

8. The isolated nucleic acid of any one of claims 1 to 7, wherein the promoter comprises a constitutive promoter, inducible promoter, or tissue- specific promoter.- 55 - #14792861vl9. The isolated nucleic acid of claim 8, wherein the promoter comprises a chicken betaactin (CBA) promoter.

10. The isolated nucleic acid of any one of claims 1 to 9, wherein the AAV ITRs are AAV2 ITRs, optionally wherein one of the AAV ITRs is a mutant ITR.

11. The isolated nucleic acid of any one of claims 1 to 10, wherein the isolated nucleic acid is located on a plasmid.

12. The isolated nucleic acid of claim 11, wherein the plasmid comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 13-21.

13. A recombinant adeno-associated virus (rAAV) comprising the isolated nucleic acid of any one of claims 1 to 10 and one or more AAV capsid proteins.

14. The rAAV of claim 13, wherein the one or more capsid proteins are AAV2 capsid proteins, or variants thereof.

15. The rAAV of claim 14, wherein the AAV2 capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 22.

16. The rAAV of claim 14 or 15, wherein the variant of the AAV2 capsid protein comprises the following amino acid substitutions relative to SEQ ID NO: 22: E36G, D80N, and VI 25 A.

17. The rAAV of claim 13, wherein the one or more AAV capsid proteins are MCI capsid proteins.

18. The rAAV of any one of claims 13 to 17, wherein the one or more AAV capsid proteins comprises the amino acid sequence set forth in SEQ ID NO: 23.-56 - #14792861vlU0120.70214WQ0019. A method for treating or preventing an ocular disease or disorder in a subject, the method comprising administering the isolated nucleic acid of any one of claims 1 to 10, or the rAAV of any one of claims 13 to 18, to a subject in need thereof.

20. The method of claim 19, wherein the ocular disease is glaucoma.- 57 - #14792861vl

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