RU2839774C2 - Engineered adeno-associated (aav) vectors for transgene expression - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is an AAV capsid protein for use in an AAV vector, wherein the AAV capsid protein contains an amino acid sequence which contains at least seven contiguous amino acids of the STTLYSP sequence (SEQ ID NO: 1). Disclosed is a nucleic acid which codes the described AAV capsid protein. Disclosed is an AAV vector for use in delivering a nucleic acid into a cell, where the AAV vector contains the described AAV capsid protein. Disclosed is a method of delivering a transgene into a cell, where the method involves bringing the cell into contact with the disclosed AAV vector.

EFFECT: invention enables to express the transgene, for example, in the central nervous system, PNS, in the inner ear, in the heart or in the retina, and also enables to express the transgene in cells of the required types.

22 cl, 9 dwg, 4 tbl, 6 ex

Description

Claims of priority

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/825,703, filed March 28, 2019. The entire contents of the foregoing are incorporated herein by reference.

Federally sponsored research or development

This invention was developed with support from the Government under Grant Nos. AG047336 and DC017117 from the National Institutes of Health. The Government has certain rights in this invention.

Field of technology

The present invention describes engineered AAV vectors for transgene expression, for example, in the CNS, PNS, inner ear, heart, or retina, and methods for using them. Methods for discovering novel engineered AAV vectors that mediate transgene expression in a desired cell type are also provided.

Prerequisites for the creation of an invention

Although AAV9 vectors have been demonstrated to have significant CNS delivery capacity following systemic administration, resulting in successful treatment of children with spinal muscular atrophy1, systemic injection of high doses of AAV vectors can result in the induction of a T cell response that can remove transduced ^cells2 . Monkeys injected with high systemic doses of an AAV9-like vector have been reported to develop toxicity resulting in death associated with systemic inflammation3 ^. A recent Phase I clinical trial using high doses of AAV9 for the treatment of muscular dystrophy was suspended by the FDA due to an immune reaction following vector infusion in one patient. The reason for requiring high doses is due to the relatively low efficiency of AAVs engineered from copies of the vector genome to ensure adequate transgene expression in a significant number of target cells. Thus, the development of new AAV capsids that would provide more efficient transduction at lower doses should provide better therapeutic efficacy while addressing safety concerns such as immunotoxicity.

The essence of the invention

The present invention describes methods that utilize an adeno-associated virus (AAV) vector genome with a two-component expression cassette to identify novel viral clones. The first component is a Cre recombinase cassette under the control of a promoter of interest. The second component is an AAV promoter for driving expression of an engineered capsid gene cloned in cis with respect to the first component of the viral genome. Viral vectors are selected for transgene expression (highly sensitive Cre expression) using cells that express a reporter gene (e.g., a green fluorescent protein) with an upstream loxP/stop site, thereby preventing reporter expression until Cre delivered by the AAV vector removes the stop site. Cells positive for the reporter gene can be isolated and the resulting AAV capsid sequences will have a higher probability of mediating efficient transgene expression. The present application also describes engineered viral sequences that provide efficient expression in the central nervous system (CNS) and peripheral nervous system (PNS), heart, liver, and inner ear.

Thus, described herein are AAV capsid proteins comprising an amino acid sequence that includes at least four contiguous amino acids of the sequence STTLYSP (SEQ ID NO: 1) or FVVGQSY (SEQ ID NO: 2). In some embodiments, the AAV capsid proteins comprise an amino acid sequence that includes at least five contiguous amino acids of the sequence STTLYSP (SEQ ID NO: 1) or FVVGQSY (SEQ ID NO: 2). In some embodiments, the AAV capsid proteins comprise an amino acid sequence that includes at least six contiguous amino acids of the sequence STTLYSP (SEQ ID NO: 1) or FVVGQSY (SEQ ID NO: 2). Alternatively, the AAV capsid proteins comprise an amino acid sequence that includes at least four, five, or six contiguous amino acids of the sequences depicted in Fig. 2A or 7C (SEQ ID NOs: 17-150).

In some embodiments, the AAV is AAV9.

In some embodiments, the AAV capsid proteins comprise AAV9 VP1.

In some embodiments, the sequence is inserted into the capsid at a position corresponding to amino acids 588 and 589 of SEQ ID NO: 6, in the VP1/VP2 border region (amino acid 138), or at any site between 583-590.

The present invention also relates to nucleic acids encoding the AAV capsid protein as described herein.

The present invention further relates to AAVs comprising the capsid proteins described herein and preferably lacking a wild-type VP1, VP2 or VP3 capsid protein. In some embodiments, the AAVs further comprise a transgene, preferably a therapeutic transgene.

The present invention further provides methods for delivering a transgene to a cell, such as to a cell in vivo or ex vivo/in vitro . These methods comprise contacting the cell with an AAV described herein. In some embodiments, the cell is a neuron (optionally a dorsal root ganglion neuron or a spiral ganglion neuron), an astrocyte, a cardiomyocyte or myocyte, an astrocyte, a glial cell, an inner hair cell, an outer hair cell, a supporting cell, an inner ear fibroblast, a photoreceptor, an interneuron, a retinal ganglion, or a retinal pigment epithelium.

In some embodiments, the cell is in a living organism, such as a mammal, and preferably a human. In some embodiments, the cell is in a tissue selected from brain, spinal cord, dorsal root ganglia, heart, inner ear, eye, or muscle, or a combination thereof. In some embodiments, the individual has Alzheimer's disease; Parkinson's disease; X-linked adrenoleukodystrophy; Canavan disease; Niemann-Pick disease; spinal muscular atrophy; Huntington's disease; connexin-26-associated disease; Usher disease type 3A; Usher disease type 2D; hair cell associated hearing loss; hair cell associated hearing loss (DFNB7/11); inner hair cell associated hearing loss (DFNB9); Usher disease type 1F; Usher disease type 1B; retinitis pigmentosa (RP; nonsyndromic); Leber congenital amaurosis; Leber hereditary optic neuropathy; Usher syndrome (RP; a syndrome associated with deafness); Duchenne muscular dystrophy; post-allograft vasculopathy; or hemophilia A and B. The methods and compositions described herein can be used to treat these conditions by administering a therapeutically effective amount of an AAV carrying a therapeutic transgene and sufficient to attenuate, reduce the risk of, or delay the onset of one or more symptoms of the condition.

In some embodiments, the cell is in the brain of an individual and the AAV is administered parenterally; intracerebrally, or intrathecally.

In some embodiments of the invention, intrathecal delivery is accomplished via lumbar injection, intracisternal injection, or intraparenchymal injection.

In some embodiments, the AAV is delivered parenterally, preferably by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection.

In some embodiments of the invention, the cell is in the eye of an individual and the AAV is administered by subretinal injection or intravitreal injection.

In some embodiments of the invention, the cell is in the inner ear of an individual and the AAV is introduced into the cochlea by application over or through the round window membrane, via a surgical cochleostomy by drilling a hole adjacent to the round window into an opening located in the bony oval window or into the semicircular canal.

The present invention also relates to a library of AAV designs comprising:

(i) the sequence encoding Cre recombinase under the control of a promoter;

(ii) a sequence encoding an AAV9 capsid protein with a peptide as described herein, such as a heptameric peptide inserted between the sequences encoding amino acids (aa) 588-589 of the capsid, under the control of a promoter located downstream of the Cre cluster. In some embodiments, the peptide comprises a randomized peptide sequence or a preselected peptide sequence.

The present invention also relates to libraries comprising a plurality of libraries of constructs described herein. In some embodiments where the peptide sequences are randomized, the library comprises constructs having sequences encoding all possible heptamer variants.

The present invention also relates to methods for identifying an engineered capsid that mediates expression of a transgene in a preselected cell type. These methods comprise: (a) administering a library according to paragraphs 23 or 24 to a non-human animal model, preferably a mammal, wherein the animal model cells express a loxP-flanked STOP cassette located upstream of a reporter sequence; (b) isolating cells of the preselected cell type; (c) selecting cells in which the reporter sequence is expressed; (d) isolating at least a portion of the library construct, preferably a portion containing a heptamer, from the selected cells in which the reporter sequence is expressed in step (c); and (e) determining the identity of the heptamers in the library constructs isolated in step (d), wherein the isolated heptamers can mediate expression of the transgene in the preselected cell type.

In some embodiments of the invention, the reporter sequence encodes a fluorescent reporter protein.

In some embodiments, the animal model is transgenic for a loxP-flanked STOP cassette located upstream of a reporter sequence, or a loxP-flanked STOP cassette located upstream of a reporter sequence may be expressed from a second construct.

In some embodiments of the invention, determining the identity of heptamers in library constructs comprises conducting DNA sequencing analysis.

In some embodiments, the methods also comprise, before and/or after step (e): performing PCR to amplify sequences comprising heptamer sequences, and optionally including full-length capsid sequences, from the library constructs isolated in step (d); cloning the amplified sequences back into a second set of library vectors; repackaging the second set of library vectors; and performing steps (a) to (d) or (a) to (e) in the second set of library vectors.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains. The methods and materials described herein are intended for use in the present invention, but other suitable methods and materials known to those skilled in the art may also be used. The materials, methods, and examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention. All publications, patent applications, patents, sequences, database entries, and other documents referenced herein are hereby incorporated by reference in their entirety. In the event of a conflict, the definitions provided in this application shall control.

Other features and advantages of the present invention will be apparent from the following detailed description and drawings, and from the claims.

Description of drawings

Fig. 1A-B. iTransduce library for selection of novel AAV capsids capable of efficiently expressing the transgene in the target tissue. a. Two-component library construction system. 1. Cre recombinase is under the control of the minimal chicken beta-actin (CBA) promoter. 2. AAV9 capsid is under the control of the p41 promoter with a randomized heptameric peptide inserted between amino acids 588-589 and cloned downstream of the Cre cluster. b. Selection strategy. i. The iTransduce library consisting of different peptide inserts expressed on the capsid (shown in different colors) was injected intravenously into Ai9 transgenic mice using a loxP-flanked STOP cluster located upstream of the tdTomato reporter gene inserted into the Gt(ROSA)26Sor locus. AAV capsids that can enter the cell of interest but do not functionally transduce the cell (no Cre expression) will not trigger tdTomato expression. Capsids that can mediate functional transduction (express Cre) will trigger tdTomato expression. ii. Cells are isolated from the organ of interest (e.g. brain) and transduced cells are selected for tdTomato expression and optionally cellular markers. iii. Capsid DNA is PCR amplified from the selected cells, cloned back into the library vector, and repackaged for another round of selection. DNA sequencing analysis is performed after each round to monitor the selection process.

Fig. 2A-B. Identification of AAV-S and AAV-F after two rounds of in vivo selection for transduction to the brain following systemic injection. Ring plots show the frequency of specific peptide insertions estimated by next-generation sequencing . a. Table of round 2 vector sequences after acquisition but before injection (SEQ ID NOs: 17-86). b. Ring plot of peptide frequencies in iTransduce isolation following round 2 injection (SEQ ID NOs 1-3). “Other” denotes sequence variants that comprise less than 1% of the total pool (in ( a ), variants isolated after round 2 screening are also highlighted in brighter color and comprise less than 1%). * denotes stop codon.

Fig. 3A-F. AAV-F efficiently enters the mouse brain after systemic injection. a. Single-chain AAV-GFP expression cassette used to compare the transduction potential of capsids. ITR, inverted terminal repeats; CBA, CMV enhancer/chicken beta-actin promoter fusion; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; pA, polyA signals (derived from both SV40 and bovine growth hormone). b . Representative low-power images of sagittal sections of whole brains of C57BL/6 mice (male) transduced with 1× ¹⁰¹¹ GV (low dose) of AAV9, AAV9-PHP.B, AAV-S, or AAV-F. c. Representative images of sagittal brain sections after injection of 8× ¹⁰¹¹ VG (high dose) of each vector in C57BL/6 males. d. Examples of spinal cord sections transduced with each of the four vectors injected intravenously at a higher dose (8× ¹⁰¹¹ VG/mouse). e. Quantification of native GFP expression from each vector as the percentage of regions covered by fluorescence at low (left panel) and high (right panel) doses. f. Multiregion comparison of transduction into the brain at the higher dose. ***, p <0.001; ****, p < 0.0001 after one-way ANOVA with Tukey's multiple comparison test (n = 3 in each group).

Fig. 4A-E. Comparison of neuronal and astrocyte transduction and biodistribution by AAV vectors. High magnification images of cells transduced with AAV9, AAV-PHP.B, AAV-S and AAV-F (GFP-positive) after co-immunostaining with markers for a. neurons (NeuN) and b. astrocytes (glutamine synthetase, GS), also shown are pooled cells with DAPI staining of the nucleus. c. Stereological assessment of the percentage of transduced cortical astrocytes and neurons after intravenous delivery of 1× ¹⁰¹¹ VG of each vector. P < 0.0001, one-way ANOVA. (*) and (**) indicate significant differences between each vector group after analysis using Tukey's multiple comparison test (n=3 mice/group). d. Biodistribution of vectors in the brain and liver assessed by qPCR of vector genomes and normalized to genomic DNA levels in the presence of GAPDH (input DNA). e. Transduction of AAV9, AAV9-PHP.B, AAV-S, and AAV-F into peripheral organs after intravenous administration of 8× ¹⁰¹¹ VG into C57BL/6 males. Retina images: RPE, retinal pigment epithelium; ONL; outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. ****, p < 0.0001 (n = 3 mice/group, one-way ANOVA with Tukey's multiple comparison test).

Fig. 5A–C. AAV-F mediates high transduction efficiency in male and female C57BL/6 mice as well as BALB/c mice. a. Representative images of GFP signal in sagittal brain sections of male and female mice (n=3) transduced with AAV-F at 1× ¹⁰¹¹ Vg/mouse. b. Sagittal brain sections of male BALB/c mice injected with AAV-F (left) or AAV9-PHP.B (right) at 1× ¹⁰¹¹ Vg/mouse. DAPI is provided as a counterstain for GFP to visualize PHP-treated brain sections. c. Quantification of endogenous GFP expression from each vector as the percentage of sections covered by fluorescence. **, p < 0.01. Unpaired t-test (n=3 mice/group).

Fig. 6A-B. AAV-F mediates higher transduction efficiency than AAV9 in human cortical neurons. a. GFP expression in human fetal primary neurons transduced with AAV-F. Neurons were labeled with anti-β-tubulin antibody to quantify transduction. b. Quantification of transduction efficiency of human neurons by AAV9, AAV-S, and AAV-F vectors. *, p < 0.05.

Fig. 7A-C. The iTransduce library functionally initiates Cre recombination, and PCR amplification of peptide-encoding 7-mer inserts in the cap gene can be performed for their isolation from tissue. a. Examples of tdTomato DAB staining in tissues after transduction with the unselected iTransduce library in Ai9 floxed/STOP tdTomato transgenic mice (right panels). PBS was included as a control (left panels). Red arrows indicate examples of transduced cells. b. Examples of PCRs maintaining the insert-containing region of the Cap gene in various tissues including the brain, compared to tissues from wild-type and transgenic non-transduced mice. c. Table illustrating the spectrum of variants observed after the first round of selection, with the most frequent variants (SEQ ID NOs: 87-150) highlighted. "Other" denotes grouped sequence variants, each of which constitutes less than 1% of the total pool. * denotes a stop codon.

Fig. 8A-B. Round 2 Cre-based selection identifies transduction-competent AAV. a. Flow cytometric analysis of tdTomato-positive cells. Following disruption of mouse brains, cell suspensions were analyzed and sorted for tdTomato-positive cells, gating based on forward and side scatter (FSC, SSC) to exclude non-viable cells (total events) and capture only individual cells (singlets), and finally for tdTomato expression (tdTomato ^+/- cells). To select tdTomato-positive cells from AAV library-transduced brains, gating was performed based on negative control (PBS-injected Ai9) and positive control (Ai9 mice transduced with AAV9-PHP.B carrying the hSyn-Cre neuron-specific cluster). b. Cre-dependent recombination events were detected by DAB immunostaining for tdTomato in the liver and in the brain after round 2 library injection (compared to PBS or AAV9-PHP.B hSyn-Cre injections). Arrow points to positive cells (astrocytes) in the brain of AAV library-injected mice.

Fig. 9A-B. Brain transduction with AAV-F and AAV-S following intravenous delivery of low (1×1011 ^VH ) or high doses of vector (8×1011 ^VH ). Transduction profile in the brain following transduction of AAV9, AAV9-PHP.B, AAV-S, and AAV-F in n=3 mice showing endogenous (unstained) GFP fluorescence in sagittal sections. Mice were injected with either 1× ¹⁰¹¹ VH (a) or 8× ¹⁰¹¹ VH (b) . Each section for each group was taken from a single mouse.

Fig. 10A-B. (a) AAV-F transduces multiple neuronal subtypes in the mouse brain. AAV-F-driven GFP expression was detected in a wide range of neuronal subtypes in different brain and CNS regions. CamKII, excitatory neurons. GAD67, inhibitory neurons. Tyrosine hydroxolase (TH), Purkinje neurons. Choline acetyltransferase (ChAT), motor neurons (white arrows represent examples of transduced neurons for each subtype). (b) AAV-F transduces efficiently, whereas AAV9 does not, at a concentration of 1×10 ¹¹ Vg/mouse. Representative 10× images of mice in Fig. 3b show GFP expression in the striatum, hippocampus, and cerebellum of AAV-F- and AAV9-injected mice.

Fig. 11. Biodistribution of AAV-F after systemic administration. Biodistribution of AAV-F compared to AAV9 is shown in skeletal muscle, heart, and spinal cord tissue. Biodistribution was assessed by qPCR of vector genomes, and samples were normalized to GAPDH genomic DNA for equal input. ns, not significant. *p < 0.05 (n=3 mice/group, each mouse sample was assessed in triplicate by a two-tailed t-test).

Fig. 12A–E. Quantification of empty capsids by transmission electron microscopy (TEM). a–d. Representative image segments of electron micrographs of AAV9 and AAV-F preparations. Two preparations of AAV9 ( a, b) and AAV-F ( c, d) were quantified by counting full and empty capsids in five images for each preparation (examples of empty capsids are indicated by arrows). e. Quantification of the percentage of empty capsid for AAV9 and AAV-F preparations. ns: not significant (p=0.54, unpaired t-test).

Fig. 13. Prolonged neuronal transduction after direct intracranial injection of AAV-F and AAV-S. Representative images of GFP (and DAPI) fluorescence signal in sagittal sections of mouse brains after direct injections of AAV-F (upper panels) or AAV-S (lower panels) into the cerebral cortex and hippocampus (1.65× ¹⁰¹⁰ and 5.6× ¹⁰¹⁰ GC/injection site for AAV-F and AAV-S, respectively). Scale bar: 1000 μm for low-magnification whole-brain images and 200 μm for high-magnification cortex and hippocampus images.

Fig. 14A-F. Broad distribution of transduction in the spinal cord and brain following lumbar intrathecal injection of AAV-F vector. Ten microliters of AAV9 (1.25 × 10 ¹¹ VG) or AAV-F (8.8 × 10 ¹⁰ VG) packaging the AAV-CBA-GFP single-chain expression cassette were injected intrathecally into the lumbar region of adult mice (n=2/vector). Three weeks later, mice were sacrificed and the spinal cord and brain were analyzed for GFP expression following immunostaining with anti-GFP antibodies. (a) Upper panels: Whole-brain coronal scans illustrating robust brain transduction by AAV-F but not AAV9. Lower panels: Whole-brain scans of spinal cord sections with AAV-F and AAV9. Mice treated with AAV-F showed very high GFP signal. AAV9 showed very low expression in both mice. All images were taken with a 33 ms exposure; an additional image was taken at 4 ms intervals for AAV-F to better resolve details. White outlines of section boundaries have been included where sections are dim. Unless otherwise noted, scale bars were 250 μm. (bd) High-magnification images of spinal cords obtained from mice treated with AAV9 (b) and AAV-F (c, d). GFAP indicates astrocyte-specific staining and NeuN indicates neurons. The spinal cord region is shown in the upper right corner of each image. Images in the lower panels are boxed images in the upper image at higher magnification. (e, f) High-magnification images of AAV-F-transduced brains after intrathecal injection. (e) Neurons in astrocytes (f) transduced by AAV-F are shown. AAV9 did not mediate detectable brain transduction following intrathecal injection. SC, spinal cord; CC, corpus callosum; CP, caudate putamen.

Fig. 15A-D. GFP fluorescence after AAV-S-CBA-GFP injection into the inner ear. (a, b) : Representative images of the cochlear sensory epithelium transduced with AAV-S (63× magnification). (c) : Transduction at the spiral limbus. ( d) : Transduction at the spiral ganglion region. Z and arrow indicate different layers of the Z-stack. OHC: outer hair cells. IHC: inner hair cells.

Fig. 16A-B. Use of the iTransduce library in non-transgenic NHPs to select AAV capsids that efficiently transduce inner ear fibrocytes and spinal cord. (A) i . Canine apes (or other non-human primates) were injected with the AAV capsid library together with AAV9-PHP.B encoding the floxed-Stop-tdTomato cluster under the control of GJB2. ii . AAV9-PHP.B will selectively express tdTomato in fibrocytes (shown by shading) if the AAV library capsid expresses Cre. iii . The inner ear is diseased. iv . tdTomato-positive fibrocytes were sorted by flow cytometry. v . Potentially functional capsids were PCR amplified from recovered DNA of sorted cells, and the library was then repackaged and subjected to another round of selection. Next-generation DNA sequencing analysis was performed after each round to monitor the selection process. Abbreviations: TM, tectorial membrane; OC, organ of Corti; SL, spiral ligament. ( B) . i . Cynomolgus monkeys (or other non-human primates) were injected with an AAV capsid library together with the AAV9-encoding floxed-Stop-mPlum cluster under the control of CBA. ii , iii . AAV9 will express mPlum in the spinal cord (shown by shading) if the AAV library capsid expresses Cre. The spinal cord is lesional and mPlum-positive cells were sorted by flow cytometry. iv . Potentially functional capsids were PCR amplified from recovered DNA of sorted cells, and the library was then repackaged and subjected to another round of selection. Next-generation DNA sequencing analysis was performed after each round to monitor the selection process.

Detailed description of the invention

A promising approach for efficient delivery of transgenes to target cells is the in vivo “survival of the fittest” selection of a pool or library of AAV vector capsid variants ^4–8 . Methods for generating AAV libraries that use randomized oligomeric nucleotides to insert short (6–9 amino acids) randomized peptides into an accessible region on the capsid surface have been shown to successfully identify novel AAV capsid variants with unique properties such as enhanced transduction of target tissues ^9,10 . One of the major limitations of AAV libraries is that the end-read of the selection process does not always distinguish capsids that mediate functional transgene expression from those that do not. AAV transduction is a multi-step process, from binding to cellular receptors and transport to the nucleus, to second-strand synthesis, and finally gene and protein expression ¹¹ . A recent refinement of a standard AAV library preparation method termed CREATE has allowed the construction of a Cre-responsive AAV genome that allows selective isolation of capsids that are successfully transported to the nucleus in transgenic animals expressing Cre ¹² . Herein, a capsid selection system is described, one example of which is iTransduce, which exploits the activity of the Cre/loxP system. Instead of using Cre transgenic mice, an AAV was engineered to encode both capsids with peptide inserts along with a Cre expression cassette. Mice were then selected with a Cre-responsive fluorescent reporter so that capsids could be selected that mediate the entire transduction process, including transgene expression. In vivo library selection identified an AAV capsid that mediates significant transduction efficiency in the CNS and another capsid that mediates transduction to the inner ear.

Using the iTransduce system, we isolated two novel AAV capsids, here termed AAV-F and AAV-S, that mediate highly efficient transgene expression in the mouse CNS (two strains tested) and inner ear, respectively. The AAV-F capsid also mediates robust transduction of human primary neurons.

Interestingly, expression-based selection identified 3 peptide clones (STTLYSP (SEQ ID NO: 1), FVVGQSY (SEQ ID NO: 2) and FQPCP* (SEQ ID NO: 3) that represented 97% of all NGS reads. Since FQPCP* has a stop codon, it was suggested that this genome was cross-packaged into another capsid during production, a phenomenon that has been reported for AAV libraries¹⁵. This may also be the case for STTLYSP (SEQ ID NO: 1, "AAV-S") (Fig. 2). AAV-S is not a defective vector because it mediates reliable transduction to peripheral organs (Fig. 4e)and into the inner ear (Fig. 15). Since high production efficiency was observed in this case (Table 4), it is possible that AAV-S may have a predisposition to cross-packaging. On the other hand, AAV-F (FVVGQSY (SEQ ID NO: 2)) was extremely efficient in transduction and was one of two potential candidates for NGS (Fig. 2). Both of these candidates were found at very low levels in the library pool in round 2 (Fig. 2), and therefore the authors were able to confirm that their enrichment was not associated with a pre-existing bias.

Since we demonstrated an iTransduce system using a cell type agnostic approach (whole brain), it is not surprising that AAV-F had a high tropism for astrocytes and neurons, i.e., cells that were transduced with AAV9. In future studies, we will combine cell type-specific promoters to drive Cre expression from an AAV library vector, as well as magnetic cell sorting to isolate capsids that can transduce cells that are refractory to traditional AAV vector transduction.

In addition to the potential of the iTransduce system to select clinical candidates for AAV vectors, it can be used to identify vectors for use as research tools. The recently identified AAV-PHP.B capsid has served as an efficient vector for genetic modification of mouse brain ¹² . However, it does not transduce BALB/c mouse lineages or BALB/c-related mice ^13,14,16 . Interestingly, robust transduction into the brain of BALB/c and C57BL/6 mice was observed following intravenous injection of AAV-F, indicating that the mechanism of enhanced transduction by AAV9 differs between AAV-PHP.B and AAV-F. This also allows AAV-F to be used as an efficient tool for CNS research in standard BALB/c mice (labome.com/method/Laboratory-Mice-and-Rats.html). In addition, as shown herein, AAV-F can also mediate sustained transgene expression in the CNS following both direct and intrathecal bolus injection, and AAV-S can mediate transgene expression in the inner ear.

Future studies in larger animals could be performed to further test AAV-F, such as in preclinical studies. Dose escalation studies could be performed to test the dose-dependent toxicity of AAV-F, as was observed for PHP.B in NHP ¹⁴ . Repeated rounds of selection could be performed in animals of different species (e.g., mice and then rats) to further ensure the efficiency of transduction between species. For example, this could be performed in mice and then rats transgenic for floxed-Stop-tdTomato ¹⁷ . Alternatively, direct selection of the iTransduce library could be performed in transgenic marmosets ^18,19 or even other non-transgenic non-human primates ( Figures 16A and B) .

Methods for identifying optimized capsid sequences

"Viral vector libraries" are pooled virus variants that, under selection pressure ( in vivo or in vitro ), can yield virus clones specific for a target cell/tissue/organ of interest. One limitation of current library generation technologies is that many of the candidate virus clones do not mediate transgene expression (the required end function of the vector). The main reason for this limitation is that no strategy has yet been developed to enable vector selection based on vector-mediated transgene expression. This application describes methods that utilize an adeno-associated virus (AAV) vector genome with an expression cassette consisting of two parts. The first part is a Cre recombinase cassette under the control of a promoter of interest. The second part is an AAV promoter to initiate expression of an engineered capsid gene cloned in cis with respect to the first part of the viral genome. Viral vectors can be selected for transgene expression (highly sensitive Cre expression) using cells that express a reporter gene (e.g., green fluorescent protein) with an upstream loxP/stop site, thereby preventing reporter expression until Cre delivered by the AAV vector removes the stop site. Cells positive for the reporter gene can be isolated and the reconstituted AAV capsid sequences will have a higher probability of mediating efficient transgene expression.

Thus, the present application describes AAV library constructs comprising: (i) a Cre recombinase under the control of a promoter, such as the minimal chicken beta-actin (CBA) promoter; (ii) an AAV9 capsid sequence under the control of a promoter (such as the p41 promoter), together with a sequence encoding a peptide described herein, such as a randomized heptameric peptide or a selected heptameric peptide, inserted into the capsid protein downstream of the Cre cluster. Preferably, the peptide is inserted between the sequences encoding amino acids (a.a.) 588-589 of the capsid, but it can also be inserted at any other site, provided that it does not negatively affect the function of the virus and maintains its activity in stimulating infection of the selected cells, for example, at the VP1/VP2 border region (a.a. 138) or at any site between amino acids 583-590. The CBA promoter is a strong active promoter that initiates Cre activity in most cell types. The P41 promoter is a natural AAV-specific promoter that initiates expression of the Cap gene. Other promoters that can be used include, but are not limited to, the synapsin promoter, the GFAP promoter, the CD68 promoter, the F4/80 promoter, the CX3CR1 promoter, the CD3 or CD4 promoter, the CMV promoter, the liver-specific promoter; and other promoters listed below. The constructs may also include a stop codon at the end of the Cre cDNA and at the end of the cap DNA. The poly-A signals are downstream of the Cre cluster and the cap cluster. Cre recombinases are known to those skilled in the art, see, e.g., Van Duyne, Microbiol Spectr. 2015 Feb; 3(1): MDNA3-0014-2014. A representative library construct is shown in Fig. 1A.

The present application also describes libraries (i.e., compositions containing a plurality of library constructs). If randomized heptameric sequences are used, it is preferred that the library include constructs with sequences encoding all or nearly all possible heptamer variants.

The methods illustrated in Fig. 1B(i) may comprise introducing a library consisting of various capsid-expressed peptide inserts (represented by various shades of grey) into a model animal, e.g., a mammal such as a mouse (e.g., an Ai9 transgenic mouse), a rabbit, a rat, or a monkey. The model animal comprises a loxP-flanked STOP cassette positioned upstream of a reporter sequence, e.g., a fluorescent reporter protein sequence, e.g., a tdTomato reporter gene, optionally inserted into the Gt(ROSA)26Sor locus. The model animal may be transgenic, or the loxP-flanked STOP cassette positioned upstream of the reporter sequence may be expressed from a second construct, e.g., a second AAV, introduced into the model animal (e.g., introduced before, during, or after introduction of the library constructs). Any AAV capsids that enter the cell of interest but do not functionally transduce the cell (express Cre) will not initiate reporter expression. Capsids that can mediate functional transduction (express Cre) will initiate tdTomato expression. As shown in Fig. 1B(ii), cells are isolated from the organ of interest (e.g., brain, eye, ear, retina, heart, etc.), and the transduced cells can then be selected for expression of the reporter gene and, optionally, cellular markers. As shown in Fig. 1B(iii), capsid DNA is obtained and analyzed, such as, but not necessarily, by PCR amplification of sequences from the sorted cells, followed by recloning back into the library vector and repackaging for another round of selection. After each round, DNA sequencing analysis can be performed to monitor the selection process.

Promoters

The library constructs described here include two promoters: one promoter drives Cre recombinase and the second drives the AAV capsid sequence.

A number of promoter sequences are known to those skilled in the art, including so-called "common" promoters that initiate expression in most cell types, such as the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the chicken beta-actin (CBA) promoter, the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the phosphoglycerol kinase promoter, the phosphoglycerol kinase (PGK) promoter, the EF1-alpha promoter, the ubiquitin C (UBC) promoter, the B-glucuronidase promoter, and the CMV immediate early/early gene enhancer/CBA promoter.

Expression of Cre recombinase can also be initiated by a tissue-specific promoter, such as a tissue-specific promoter for the CNS, liver, heart, cochlea, retina, or T cells, inter alia . In some embodiments, a tissue-specific promoter for the CNS includes a neuronal, macrophage/microglial cell, and astrocyte promoters. A number of tissue-specific promoters are known to those skilled in the art, including the synapsin promoter (neurons), neuron-specific enolase (NSE) (neurons), MeCP2 (methyl-CPG-binding protein 2) (neurons), glial fibrillary acidic protein (GFAP) (astrocytes), oligodendrocyte transcription factor 1 (Olig1) (oligodendrocytes), CNP (2',3'-cyclic nucleotide-3'-phosphodiesterase) (broad-spectrum cells), or CBh (a hybrid promoter of CBA or the MVM intron and CBA) (broad-spectrum cells). See, for example, U.S. Patent No. 20190032078. Macrophage/microglial cell promoters include, but are not limited to, the C-X3-C motif chemokine receptor 1 (CX3CR1) promoter, the CD68 promoter, the calcium ion binding adaptor molecule 1 (IBA1) promoter, the transmembrane protein 119 (TMEM119) promoter, the spalt-like transcription factor 1 (SALL1) promoter, the G protein-coupled adhesion receptor E1 (F4/80) promoter, the myeloproliferative sarcoma virus enhancer, the d1587rev primer binding site swapped negative control region deleted (MND) promoter; integrin alpha-M subunit promoter (ITGAM; CD11b-myeloid cell (neutrophil, monocyte, and macrophage) promoter). For expression in the inner ear, the promoter may be, for example, PKG, CAG, prestin, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, oncomod, or the myo7A promoter; see Ryan et al., Adv Otorhinolaryngol. 2009; 66: 99-115.

Reporter proteins

A number of reporter proteins are known to those skilled in the art, including green fluorescent protein (GFP), green fluorescent protein variant (GFP10), activated GFP (eGFP), TurboGFP, GFPS65T, TagGFP2, mUKGEmerald GFP, Superfolder GFP, GFPuv, destabilized EGFP (dEGFP), Azami Green, mWasabi, Clover, mClover3, mNeonGreen, NowGFP, Sapphire, T-Sapphire, mAmetrine, photoactivatable GFP (PA-GFP), Kaede, Kikume, mKikGR, tdEos, Dendra2, mEosFP2, Dronpa, blue fluorescent protein (BFP), eBFP2, azurite BFP, mTagBFP, mKalamal, mTagBFP2, shBFP, Blue Fluorescent Protein (CFP), eCFP, Cerulian CFP, SCFP3A, Destabilized ECFP (dECFP), CyPet, mTurquoise, mTurquoise2, mTFP1, Photoswitchable CFP2 (PS-CFP2), TagCFP, mTFP1, mMidoriishi-Cyan, Aquamarine, mKeima, mBeRFP, LSS-mKate2, LSS-mKatel, LSS-mOrange, CyOFP1, Sandercyanin, Red Fluorescent Protein (RFP), eRFP, mRaspberry, mRuby, mApple, mCardinal, mStable, mMaroonl, mGarnet2, tdTomato, mTangerine, mStrawberry, TagRFP, TagRFP657, TagRFP675, mKate2, HcRed, t-HcRed, HcRed-Tandem, mPlum, mNeptune, NirFP, Kindling, far-red fluorescent protein, yellow fluorescent protein (YFP), eYFP, destabilized EYFP (dEYFP), TagYFP, Topaz, Venus, SYFP2, mCherry, PA-mCherry, Citrine, mCitrine, Ypet, IANRFP-AS83, mPapayal, mCyRFP1, mHoneydew, mBanana, mOrange, Kusabira Orange, Kusabira Orange 2, mKusabira Orange, mOrange 2, mKO _K , mKO2, mGrapel, mGrape2, zsYellow, eqFP611, Sirius, Sandercyanin, shBFP-N158S/L173I, protein, near infrared fluorescent, iFP1.4, iRFP713, iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, miRFP670, brilliant violet (BV) 421, BV 605, BV 510, BV 711, BV786, PerCP, PerCP/Cy5.5, DsRed, DsRed2, mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, or phycobiliprotein, or a biologically active variant or fragment of any of them.

Sets

The present invention also relates to kits described herein comprising one or more AAV library constructs with or without randomized heptameric sequences. The kits may also include a construct comprising a loxP-flanked STOP cassette located upstream of a reporter sequence.

Engineered AAV capsid proteins

The methods of the invention identify two peptide sequences that alter the ability of an AAV to mediate transgene expression in certain cells when incorporated into the capsid of an AAV, such as AAV1, AAV2, AAV8, or AAV9. In some embodiments, the peptides comprise sequences of at least 7 amino acids. In some embodiments, the amino acid sequence comprises at least 4, such as 5, 6, or 7 contiguous amino acid sequences (STTLYSP (SEQ ID NO: 1) or FVVGQSY (SEQ ID NO: 2).

Peptides comprising reverse sequences, such as PSYLTTS (SEQ ID NO: 4) and YSQGVVF (SEQ ID NO: 5), may also be used. Alternatively, the peptides may comprise at least four, five or six contiguous amino acids of the sequences shown in Fig. 2A or 7C (SEQ ID NOs: 17-150).

AAV

Viral vectors for use in the methods, kits and compositions of the invention include recombinant retroviruses, adenovirus, adeno-associated virus, alphavirus and lentivirus, preferably comprising a capsid peptide as described herein and optionally a transgene for expression in a target tissue.

A preferred viral vector system useful for delivering nucleic acids in the methods of the invention is adeno-associated virus (AAV). AAV is a small non-enveloped virus having a 25 nm capsid. No disease has been known or reported to be associated with this wild-type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-lasting episomal transgene expression and excellent transgene expression in the brain, particularly in neurons. The space for exogenous DNA is limited to approximately 4.7 kb. An AAV vector, such as that described in Tratschin et al., Mol. Cell. Biol. 5: 3251-3260 (1985), can be used to introduce DNA into cells. Various nucleic acids have been introduced into various cell types using AAV vectors (see, e.g., Hermonat et al., Proc. Natl. Acad. Sci. USA 81: 6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4: 2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2: 32-39 (1988); Tratschin et al., J. Virol. 51: 611-619 (1984); and Flotte et al., J. Biol. Chem. 268: 3781-3790 (1993). Many alternative AAV variants exist (more than 100 have been cloned), and AAV variants have been identified based on desired properties. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, rh.8, AAV9, rh.10, rh.39, rh.43, or CSp3; and for use in the CNS, in some embodiments, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As one example, AAV9 has been shown to be somewhat effective at penetrating the blood-brain barrier. Using the methods of the invention, the AAV capsid can be genetically modified to enhance the ability to penetrate the BBB, or a particular tissue, by inserting a peptide sequence described herein into a capsid protein, such as into the AAV9 capsid protein VP1 between amino acids 588 and 589.

A representative sequence of the wild-type AAV9 capsid protein VP1 (Q6JC40-1) is:

Thus, the present invention relates to AAVs that comprise one or more peptide sequences described herein, for example, an AAV comprising a capsid protein comprising a sequence described herein, for example, a capsid protein comprising SEQ ID NO: 1 or SEQ ID NO: 2, wherein a peptide sequence has been inserted into the sequence, for example, between amino acids 588 and 589.

Representative AAV sequences are shown below. Inserted peptide sequences are shown in bold and double underlined in protein sequences, and bold and capitalized in DNA sequences.

AAV-F capsid protein sequence

FVVGQSY AQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO:7)

AAV-F capsid DNA sequence

TTTGTTGTTGGTCAGAGTTAT gcacaggcgcagaccggctgggttcaaaaccaaggaatacttccgggtatggtttggcaggacagagatgtgtacctgcaaggacccatttgggccaaaattcctcacacg gacggcaactttcacccttctccgctgatgggagggtttggaatgaagcacccgcctcctcagatcctcatcaaaaacacacctgtacctgcggatcctccaacggccttc aacaaggacaagctgaactctttcatcacccagtattctactggccaagtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaagcgctggaacccggagatc cagtacacttccaactattacaagtctaataatgttgaatttgctgttaatactgaaggtgtatatagtgaaccccgccccattggcaccagatacctgactcgtaatctg (SEQ ID NO:8)

AAV-S capsid protein sequence

STTLYSP AQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO:9)

Capsid DNA sequence AAV-S

TCTACTACGCTTTATAGTCCT gcacaggcgcagaccggctgggttcaaaaccaaggaatacttccgggtatggtttggcaggacagagatgtgtacctgcaaggacccatttgggccaaaattcctcacacg gacggcaactttcacccttctccgctgatgggagggtttggaatgaagcacccgcctcctcagatcctcatcaaaaacacacctgtacctgcggatcctccaacggccttc aacaaggacaagctgaactctttcatcacccagtattctactggccaagtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaagcgctggaacccggagatc cagtacacttccaactattacaagtctaataatgttgaatttgctgttaatactgaaggtgtatatagtgaaccccgccccattggcaccagatacctgactcgtaatctg (SEQ ID NO:10).

The AAV sequences may be, for example, at least 80, 85, 90, 95, 97 or 99% identical to an AAV reference sequence provided herein, and, for example, may preferably include variants that do not reduce the ability of the AAV to mediate expression of a transgene in a cell. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned to optimally compare them (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences to optimally align, wherein non-homologous sequences may be ignored in the comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, the term "identity" of amino acids or nucleic acids is equivalent to the term "homology" of amino acids or nucleic acids). The percentage of identity between two sequences depends on the number of identical positions in both sequences, as well as the number of gaps and the length of each gap that must be introduced to optimally align the two sequences.

Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48: 444–453) algorithm, which has been incorporated into the GAP program in the GCG software package (available online at gcg.com), using default parameters, such as the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a gap-frameshift penalty of 5.

Transgenes

In some embodiments, the AAV also includes a transgene sequence (i.e., a heterologous sequence), e.g., a transgene encoding a therapeutic agent, e.g., as described herein or known to those skilled in the art, or a reporter protein, e.g., a fluorescent protein, an enzyme that catalyzes a reaction to produce a detectable product, or a cell surface antigen. The transgene is preferably linked to sequences that promote/initiate expression of the transgene in the target tissue.

Examples of transgenes for use as therapeutic agents include neuronal apoptosis inhibitory protein (NAIP); nerve cell growth factor (NGF); glial-derived growth factor (GDNF); brain-derived growth factor (BDNF); ciliary neurotrophic factor (CNTF); tyrosine hydroxylase (TH); GTP cyclohydrolase (GTPCH); amino acid decarboxylase (AADC); aspartoacylase (ASPA); blood factors such as β-globin, hemoglobin, tissue plasminogen activator and coagulation factors; colony stimulating factors (CSF); interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc. growth factors such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGF), bone morphogenesis protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma-derived growth factor (HDGF), myostatin (GDF-8), nerve cell growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), etc.; soluble receptors such as soluble TNF-α receptors, soluble VEGF receptors, soluble interleukin receptors (e.g. soluble IL-1 receptors and soluble IL-1 type II receptors), soluble T-cell receptors gamma/delta, soluble receptor ligand-binding fragments, etc.; enzymes such as α-glucosidase, imiglucarase and β-glucocerebrosidase; enzyme activators such as tissue plasminogen activator; chemokines such as IP-10, monokine induced by gamma interferon (Mig), Groa/IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4, etc.; angiogenic agents such as vascular endothelial growth factors (VEGF, such as VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor beta, basic fibroblast growth factor, glioma-derived growth factor, angiogenin, angiogenin-2, etc.; antiangiogenic agents such as soluble VEGF receptor; protein vaccine; Neuroactive peptides such as nerve growth factor (NGF), bradykinin, cholecystokinin, hastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing factor, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, silencing peptide, etc.; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); transforming growth factors (TGF); tissue factors, luteinizing hormone; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptor antagonists; etc. Some other examples of protein of interest include ciliary neurotrophic factor (CNTF); neurotrophins 3 and 4/5 (NT-3 and 4/5); glial cell-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia-associated coagulation proteins such as factor VIII, factor IX, factor X; dystrophin or ninidystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); enzymes associated with glycogen storage disease such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N-acetylhexosaminidase A); and any variants thereof.

The transgene may also encode an antibody, such as an immune checkpoint inhibitory antibody such as an antibody against PD-L1, PD-1, CTLA-4 (cytotoxic T lymphocyte-associated protein 4; CD152); LAG-3 (lymphocyte activation gene 3; CD223); TIM-3 (T-cell immunoglobulin and mucin domain 3; HAVCR2); TIGIT (T-cell immunoreceptor with Ig and ITIM domains); B7-H3 (CD276); VSIR (V-series immunoregulatory receptor, also known as VISTA, B7H5, C10orf54); BTLA 30 (B and T lymphocyte attenuator, CD272); GARP (glycoprotein A domains with large number of repeats); PVRIG (PVR-associated immunoglobulin domain-containing protein); or VTCN1 (V series domain-containing T cell activation inhibitor 1, also known as B7-H4).

Other transgenes may include small or inhibitory nucleic acids that alter/reduce the expression of a target gene, such as siRNAs, shRNAs, miRNAs, antisense oligonucleotides or long non-coding RNAs that alter gene expression (see, for example, WO2012087983 and US20140142160), or CRISPR Cas9/cas12a and guide RNAs.

The virus may also include one or more sequences that promote expression of the transgene, such as one or more promoter sequences; enhancer sequences, such as a 5' untranslated region (UTR) or 3' UTR; a polyadenylation site; and/or insulator sequences. In some embodiments, the promoter is a brain tissue-specific promoter, such as a neuron-specific promoter or a glial cell-specific promoter. In some embodiments, the promoter is a gene promoter selected from the neuronal nuclei promoter (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous intestinal polyposis (APC), calcium ion-binding adaptor molecule 1 (Iba-1), synapsin I (SYN), calcium/calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase, and platelet-derived growth factor beta chain. In some embodiments, the promoter is a cell type promoter, such as the cytomegalovirus (CMV), beta-glucuronidase (GUSB), ubiquitin C (UBC), or Rous sarcoma virus (RSV) promoter. The woodchuck hepatitis virus post-transcriptional response element (WPRE) can also be used.

In some embodiments, the AAV also has one or more additional mutations that improve delivery to a target tissue, such as the CNS, or decrease targeting to non-target tissue, such as mutations that decrease delivery to the liver when delivery to the CNS, heart, or muscle is desired (e.g., as described by Pulicherla et al. (2011) Mol Ther 19: 1070-1078); or the addition of other peptides, such as described by Chen et al. (2008) Nat Med 15: 1215-1218 or Xu et al., (2005) Virology 341: 203-214 or U.S. Pat. Nos. 9,102,949; 9585971 and 20170166926. See also Gray and Samulski (2011) “Vector design and considerations for CNS applications” in Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorioso J., editor. (Washington, DC: Society for Neuroscience) 1-9, available at sfn.org/~/media/SfN/Documents/Short%20Courses/ 2011%20Short%20Course%20I/2011_SC1_Gray.ashx.

Methods of application

The methods and compositions described herein can be used to deliver any composition, such as a sequence of interest, to a tissue, such as the central nervous system (brain), heart, muscle, peripheral nervous system (e.g., dorsal root ganglia or spinal cord), or inner ear or retina. In some embodiments, the methods include delivery to specific areas of the brain, such as the cerebral cortex, cerebellum, hippocampus, substantia nigra, tonsils. In some embodiments, the methods include delivery to the lumbar region, such as the subarachnoid space or the epidural space. In some embodiments, the methods include delivery to neurons, astrocytes, or glial cells. In some embodiments, the methods include delivery to inner and/or outer hair cells, spiral ganglion neurons, supporting cells, or inner ear fibrocytes. In some embodiments, the methods include delivery to photoreceptors, interneurons, retinal ganglion cells (e.g., using AAV-F), or retinal pigment epithelium (RPE) (e.g., using AAV-S).

In some embodiments, the methods and compositions, e.g., AAV, are used to deliver a nucleic acid sequence to an individual suffering from a disease, e.g., a CNS disease; see, e.g., U.S. Patents 9,102,949; 9,585,971; and U.S. Patent Application 20170166926. In some embodiments, the individual suffers from the diseases listed in Tables 1-3; and in some embodiments, the vectors are used to deliver a therapeutic agent listed in Tables 1-3 to treat the corresponding diseases listed in Tables 1-3. The therapeutic agent can be delivered as a nucleic acid, e.g., via a viral vector, wherein the nucleic acid encodes a therapeutic protein or another nucleic acid such as an antisense oligonucleotide, siRNA, shRNA, etc.; or as a fusion protein/complex with a peptide as described herein.

The methods and compositions described herein can be used to treat these conditions in an individual in need thereof by administering a therapeutically effective amount of an AAV carrying a therapeutic transgene, namely, an amount sufficient to alleviate, reduce the risk of, or delay the onset of one or more symptoms of the condition.

Table 1. CNS targets (AAV-F, AAV-S)

Disease Target genes Target cells/tissues Link Alzheimer's disease CD33, APOE, BACE, Brain (Griciuc et al., 2013) Parkinson's disease GDNF, AADC Brain
Neurons (Christine et al., 2019) X-linked adrenoleukodystrophy ABCD1 Brain/Spinal Cord Neurons/Astrocytes/Microglia/Endothelial Cells (Eichler et al., 2017; Gong et al., 2015) Canavan disease ASPA Brain (Leone et al., 2012) Niemann-Pick disease NPC1 Brain (Hughes et al., 2018) Spinal muscular atrophy SMN Motor neurons (Al-Zaidy et al., 2019) Huntington's disease HTT Neurons of the brain (Caron et al., 2020; Keskin et al., 2019)

Table 2. Inner Ear Targets (AAV-S)

Disease Target genes Target cells/tissues Link Connexin 26 disease GJB2 Inner ear - cochlea
Fibrocytes/support cells ARO 2020 Usher disease type 3A* CLRN1 Inner ear - cochlea
Hair cells (inner and outer) (György et al., 2019) Usher disease type 2D* WHLN Inner ear - cochlea
Hair cells (inner and outer) (Isgrig et al., 2017) Hair cell hearing loss ATOH1 Inner ear - cochlea
Support cells (for HC regeneration) (Tan et al. 2019) Hair cell-mediated hearing loss (DFNB7/11) TMC1 Inner ear - cochlea
Hair cells (inner and outer) (Nist-Lund et al., 2019) Inner hair cell-mediated hearing loss (DFNB9) OTOF Inner ear - cochlea
Inner hair cells (Аkil et al., 2019) Usher disease type 1F* PCDH15 Inner ear - cochlea
Hair cells (inner and outer) Review (Zhang et al., 2018) Usher disease type 1B* Myo7a Inner ear - cochlea
Hair cells (inner and outer) Review (Lopes and Williams, 2015)

* Usher syndrome results in both deafness, as noted above, and blindness due to retinitis pigmentosa.

Table 3. Targets for the peripheral system (AAV-F, AAV-S)

Diseases Target genes Target cells/tissues Link Retinitis pigmentosa (RP; nonsyndromic) RHO, RPGR, RP2, NRL and others Retina (photoreceptors) (Cehajic-Kapetanovic et al., 2020; Millington-Ward et al., 2011; Mookherjee et al., 2015; Yu et al., 2017) Leber's congenital amaurosis RPE65 Retina (retinal pigment epithelium) (Maguire et al., 2019) Leber's hereditary optic neuropathy ND1-6 (mitochondrial) Retina (retinal nodule cells) (Wan et al., 2016; Yang et al., 2016) Usher syndrome (RP; syndrome with deafness) Genes that cause Usher disease and are listed above Retina (photoreceptors) As stated above Duchenne muscular dystrophy Dystrophin Muscles Review (Duan, 2018) Vasculopathy after allotransplantation TIMP-1 Heart (Remes et al., 2020) Hemophilia A and B FVIII, FIX Liver Review (Nathwani, 2019)

Pharmaceutical compositions and routes of administration

The methods described herein include the use of pharmaceutical compositions comprising AAV as an active ingredient.

Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and agents delaying absorption, and the like, compatible with the administration of pharmaceuticals.

Pharmaceutical compositions are typically formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or by injection or infusion. Delivery may thus be systemic or local. For example, delivery to the inner ear may involve administration into the cochlea over or through the round window membrane, via a surgical cochleostomy by drilling a hole adjacent to the round window, via an opening in the oval window of bone, or via the semicircular canal. (See, e.g., Kim et al., Mol Ther Methods Clin Dev. 2019 Jan 11: 13: 197-204; Ren et al., Front Cell Neurosci. 2019; 13: 323); and for delivery to the retina, subretinal or intravitreal injections can be administered (see, e.g., Ochakovski et al., Front Neurosci. 2017; 11: 174; Xue et al., Eye (Lond). 2017 Sep; 31 (9): 1308–1316).

Methods for preparing suitable pharmaceutical compositions are known to those skilled in the art, see, for example, Remington: The Science and Practice of Pharmacy , 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). Thus, for example, solutions or suspensions used for parenteral administration can include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The preparation for parenteral administration may be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders prepared as sterile solutions or dispersions for extemporaneous administration by injection. For intravenous administration, suitable carriers are saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that it can be easily withdrawn by syringe. 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, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyhydric alcohols such as mannitol and sorbitol; and sodium chloride. Prolonged absorption of injection compositions can be achieved by including in the composition an agent retarding absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle which contains the basic dispersion medium and the required other ingredients from among 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, whereby a powder consisting of the active ingredient and any additional desired ingredient is obtained from the solution by sterile filtration.

In one embodiment of the invention, the therapeutic compounds are prepared together with carriers that will protect the therapeutic compounds from rapid elimination from the body, for example, in the form of a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Such formulations can be prepared by standard methods or they can be purchased from, for example, Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells by monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared by methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

The pharmaceutical compositions may be included in a kit, container, package, or dispenser along with instructions for use. For example, a kit may include compositions comprising an AAV comprising a peptide as described herein.

EXAMPLES

The present invention is further described in the following examples, which should not be construed as limiting the scope of the invention as set forth in the claims.

Materials and methods

Unless otherwise noted, the following materials and methods were used in the examples below.

Construction of the AAV library.

iTransduce Plasmid: pAAV-CBA-Cre ^mut -p41-Cap9del

We constructed an iTransduce library backbone plasmid, named pAAV-CBA-Cre ^mut -p41-Cap9del, containing two expression cassettes in cis orientation: 1) CBA-Cre ^mut , into which we introduced a mutant Cre cDNA (CCG → CCT, encoding the Pro15 amino acid to remove the initially present AgeI site) under the control of the common CBA promoter, 2) p41-Cap9del, consisting of the AAV9 capsid gene under the control of the AAV5 p41 promoter (residues 1680-1974 GenBank AF085716.1) and the splicing sequence of the AAV2 rep gene (similar to the gene described with reference ¹² ).

A mutant Cre cDNA (Cre ^mut ) flanked by KpnI and SalI restriction sites and a p41-CAP9(del)-polyA fragment were synthesized using GenScript and cloned into the puC57 backbone. We first constructed the plasmid pAAV-CBA-Cre ^mut -polyA by subcloning the mutant Cre cDNA (with the KpnI-blunt, SalI fragment) in place of the eGFP-WPRE into the inventive pAAV-CBA-WPRE backbone (linearized with NcoI-blunt/SalI, which removes the originally present eGFP-WPRE fragment). We then introduced the p41-Cap9del fragment carrying the K449R mutation in the Cap9 sequence, creating a unique XbaI site where the 447 bp Cap sequence was deleted. between the restriction sites XbaI and AgeI.

Plasmid for generation of randomized 7-mer peptide sar fragments and subcloning of CAP9 fragments obtained by PCR: pUC57-Cap9-XbaI/KpnI/AgeI.

We first obtained the 447 bp region of the AAV9 capsid sequence between the XbaI/AgeI sites (a sequence that is absent from pAAV-CBA-Cre ^mut -p41-Cap9del), synthesized by Genscript (Piscataway, NJ) and subcloned into pUC57-Kan. This capsid sequence has the same deletion of the EarI site in this 447 bp region of the wild-type AAV9 cap, allowing removal of the restriction digest from wild-type AAV9 as described by Deverman et al. 2015. A unique KpnI site was also created. The pUC57-Cap9-XbaI/KpnI/AgeI cap fragment was used to generate a starting library of randomized 21-mer nucleotide sequences (encoding 7-mer peptides) inserted between the nucleotides encoding amino acids 588 and 589 of AAV9 VP1. We used a strategy similar to that described by Deverman et al. (2015). Briefly, pUC57-Cap9-XbaI/KpnI/AgeI served as a template for cap DNA amplification and insertion of randomized 21-mer sequences using forward and reverse primers. Primer information: XF-extended primer (5'-GTACTATCTCTCTAGAACtattaacggttc-3'; SEQ ID NO: 11) and reverse primer 588iRev 5'-(GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCXMNNMNNMNNMNNMNNMN

NMNNTTGGGCACTCTGGTGGTTTGTG-3'; SEQ ID NO: 12), where INN repeat stands for randomized 21-mer nucleotides (purchased from IDT). XF-extended primer and 588iRev were used in PCR using Phusion polymerase ( NEB) and pUC57-Cap9-XbaI/KpnI/AgeI as a template. The 447 bp PCR product was digested with XbaI and AgeI overnight at 37°C and then gel purified (Qiagen). Similarly, pAAV-CBA-Cre ^mut -p41-Cap9del was digested with XbaI and AgeI and gel purified. A ligation reaction was then performed (1 h at room temperature) with T4 DNA ligase (NEB) using a molar ratio of 3:1 cap insert to vector. The ligated plasmid was then named pAAV-CBA-Cre ^mut -p41-Cap9-7mer, and this plasmid contained a pool of plasmids with randomized 7-mer peptides inserted into the cap gene between nucleotides encoding 588 and 589 of AAV9 VP1.

This plasmid ( pUC57-Cap9-XbaI/KpnI/AgeI) was also used as the recipient plasmid of the invention for subcloning CAP9 fragments amplified by PCR from brain tissue. We removed the upstream KpnI site in pUC57 by digestion with SacI and NsiI and ligation. This created a unique KpnI site in the capsid fragment. See below.

Plasmid for expression of rep.

We constructed a rep expression plasmid, named pAR9-Cap9-stop/AAP/Rep, using a strategy similar to that described by Deverman et al. ¹² . Full-length cDNA was synthesized using Genscript and cloned into pUC57-Kan. Stop codons were inserted in place of the start codons for VP1, VP2, and VP3 so that no parental AAV9 capsids were produced but AAP and rep expression was maintained.

Creating and cleaning an AAV library.

For each production, we seeded 15-cm tissue culture dishes with 1.5 × 10 ⁷ 293T cells/dish. The next day, the cells were transfected with adenovirus helper plasmid (pAdΔF6, 26 μg/dish), rep plasmid (pAR9-Cap9-stop/AAP/Rep, 12 μg/dish), and ITR-flanked AAV library (pAAV-CBA-Cre-mut/p41-Cap9-7mer, 1 μg/dish) by the calcium phosphate method to induce AAV production. The day after transfection, the medium was replaced with DMEM containing 2% FBS. AAV was purified from the cell lysate by iodixanol density gradient ultracentrifugation. Buffer exchange to PBS was performed using ZEBA spin columns (7K MWCO; Thermo Fisher Scientific) and subsequent concentration was performed on Amicon Ultra 100 kDa MWCO ultrafiltration centrifugal devices (Millipore). Vectors were stored at -80°C until use. We then quantified AAV genomic copies (AG) in AAV preparations by TaqMan qPCR using ITR sequence-specific primers and probes ^20,21 .

Next generation library sequencing.

Next-generation sequencing was performed on a pooled AAV9 plasmid library, followed by capsid packaging. Sequencing was also performed after PCR recovery of the cap fragment (either from brain tissue or from isolated tdTomato-positive cells selected by flow cytometry). For each round of selection, viral DNA corresponding to the region containing the insert was PCR amplified using the Phusion High-Fidelity PCR kit from New England Biolabs (forward primer: 5′-AATCCTGGACCTGCTATGGC-3′ (SEQ ID NO: 13), reverse primer: 5′-TGCCAAACCATACCCGGAAG-3′ (SEQ ID NO: 14)). PCR amplification was performed using Q5 polymerase (New England Biolabs). Unique barcode adapters were hybridized to each sample and the samples were sequenced on an Illumina Miseq (150 bp reads). Approximately 50-100,000 reads per sample were analyzed. The resulting sequence files were qualitatively verified using FastQC (bioinformatics.babraham.ac.uk/projects/fastqc/) and analyzed using a program written specifically in Python. Briefly, sequences were quantitatively analyzed for the presence or absence of an insert, sequences containing an insert were then compared to a base reference sequence, and error-free reads were tabulated based on the occurrence of each unique insert detected. Inserts were translated and normalized.

Animals.

All animal experiments were approved by the Massachusetts General Hospital Animal Care Subcommittee in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. We used adult (8-10 weeks old) Ai9 (type #007909), C57BL/6 (type #000664), and BALB/c (type #000651) mice from The Jackson Laboratory, Bare Harbor, Maine. All animals were euthanized three weeks after injection, transcardially perfused, and tissues were collected and either fixed in 4% paraformaldehyde in PBS and snap-frozen in liquid nitrogen or dissociated for flow cytometry.

In vivo selection of capsids with tropism for the brain.

Ai9 mice were injected intravenously (tail vein) with the dose of VG indicated in the Results section, and 3 weeks after injection, the mice were sacrificed and tissues were collected.

Mice were deeply anesthetized with isofluorane and decapitated. In round 1, brains were rapidly dissected and two coronal sections (2 mm thick) were taken. One section was used for full-length brain DNA extraction (DNeasy Blood and Tissue Kits, Qiagen, Hilden, Germany). The other coronal section was fixed in 4% PFA and embedded in paraffin for immunohistological analysis (tdtomato-positive cells were detected after each round of selection by DAB staining using rabbit anti-RFP antibody from Rockland Immunochemicals). For round 2, brain tissue was then cut into 1 ^mm3 pieces with a razor blade and neural cells were isolated by papain dissociation (Papain Dissociation System, Worthington) according to the manufacturer's instructions. Following dissociation, myelin was removed (using Miltenyi Human, Mouse, and Rat Myelin II Removal Beads) and tdTomato-positive cells were sorted using an S3eTM Cell Sorter (Bio-Rad). Cells were sorted by a first series of gating to exclude cell debris and select only singlets. Cell suspensions from AAV9-PHP.B-Cre-injected Ai9 mice (positive control) and PBS-injected Ai9 mice (negative control) were used to perform a series of gating to select tdTomato-positive and negative cells. Following selection, tdTomato-positive cells were immediately pelleted by centrifugation and DNA was extracted using the ARCTURUS PicoPure DNA Extraction Kit (ThermoFisher).

Following DNA extraction, Cap9 inserts (containing the 21-mer sequence encoding 7-mer peptides) were amplified using the following primers: Cap9_Kpn/Age_For: 5'-AGCTACCGACAACAACGTGT-3' (SEQ ID NO: 15) and Cap9_Kpn/Age_Rev: 5'-AGAAGGGTGAAAGTTGCCGT-3' (SEQ ID NO: 16) (using the Phusion High Confidence PCR Kit, New England Biolabs). The amplicons were then purified (with the Monarch PCR and DNA Purification Kit, New England Biolabs), digested with KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were purified on agarose gel (with the Monarch DNA Gel Extraction Kit, New England Biolabs) followed by ligation into the plasmid pUC57-Cap9-XbaI/AgeI/KpnI (linearized with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). The ligation products were transformed into electrocompetent DH5alpha bacteria (New England Biolabs), and all transformed bacteria were grown overnight in LB medium with ampicillin. Plasmid pUC57-Cap9-XbaI/AgeI/KpnI was purified using the Maxi-Prep kit (Qiagen). The plasmid was digested with XbaI/AgeI to release the 447 bp cap fragment, which was then gel purified and ligated with similarly cut pAAV-CBA-Cre-mut/p41-Cap9del for the next round of AAV library preparation.

AAV rep/cap plasmids containing AAV-F and AAV-S peptide inserts for vector generation.

To generate rep/cap plasmids encoding AAV9 capsids carrying a peptide insert of interest for production of vectors encoding a transgene of interest (e.g., GFP), we digested the AAV9 rep/cap plasmid with BsiWI and BaeI, which remove the fragment flanking amino acid 588 of VP3 at the insertion site for the peptide sequence. We then ordered from Integrated DNA Technologies (IDT, Coralville, IA) a 997-bp dsDNA fragment that contains overlapping Gibson homology arms to BsiWI/BaeI-cut AAV9, as well as a 21-mer nucleotide sequence encoding the peptide of interest in frame after amino acid 588 of VP3. Finally, we performed Gibson assembly using Gibson Assembly® Master Mix (NEB, Ipswich, MA) to ligate the peptide-containing insert into the AAV9 rep/cap plasmid.

AAV vectors for transduction analysis.

For each production, we seeded 15-cm tissue culture dishes with 1.5 × 10 ⁷ 293T cells/dish. The next day, the cells were transfected with an adenovirus helper plasmid (pAdΔF6, 26 μg/dish), a rep/cap plasmid (AAV9, AAV-F, AAV-S;, 12 μg/dish), and an ITR-flanked transgene cassette plasmid (single-stranded AAV-CBA-GFP-WPRE ²² , 10 μg/dish) to induce AAV production. The day after transfection, the medium was replaced with DMEM containing 2% FBS. AAV was purified from the cell lysate by iodixanol density gradient ultracentrifugation. Buffer exchange to PBS was performed using ZEBA spin columns (7K MWCO; Thermo Fisher Scientific) and subsequent concentration was performed on Amicon Ultra 100 kDa MWCO ultrafiltration centrifugal devices (Millipore). Vectors were stored at -80°C until use. We then quantified AAV genomic copies in AAV preparations by TaqMan qPCR using primers and probes specific for the BGH-polyA sequence ²³ .

Animal euthanasia and tissue collection

Mice (strain indicated in each figure) were slowly injected via the lateral tail vein with 200 μl of the AAV test vector diluted in sterile PBS (low dose: 4 × 10 ¹² VG/kg and high dose: 3.2 × 10 ¹² VG/kg) using gentle finger pressure at the injection site until bleeding stopped. Three weeks after injection, mice were euthanized and transcardially perfused with sterile cold phosphate-buffered saline (PBS). The brain was then longitudinally dissected into two hemispheres. One hemisphere was post-fixed in 15% glycerol/4% paraformaldehyde diluted in PBS for 48 h and then in 30% glycerol for cryopreservation for an additional 48–72 h. For the high dose group, a small piece of heart, muscle (gastrocnemius) and retina were also processed for immunohistological analysis. We obtained three independent preparations of AAV-S, AAV-F and AAV9 (Table I). The transduction results in mice were obtained from one preparation of each vector, however, we reproduced these results in two more independent experiments.

Immunohistology and high magnification imaging of AAV-CBA-GFP transduced neural and neuronal cell populations

Coronal floating sections (40 μm) were cut using a microtome-cryostat. After washing off the glycerol in Tris-buffered saline (TBS), cryosections were permeabilized with 0.5% Triton X-100 (AmericanBio) in TBS for 30 min at room temperature, and blocked with 5% normal goat serum (or normal donkey serum) and 0.05% Triton in TBS for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C in 2.5% NGS and 0.05% Triton in TBS, and secondary antibodies conjugated with Alexa Fluor 488 or Cy3 (Jackson ImmunoResearch laboratories, Baltimore, USA) were incubated for 1 h the following day. The primary antibodies used in this study were: chicken anti-GFP antibody (Aves Labs, Tigard, USA), mouse anti-NeuN antibody (EMD Millipore, Burlington, USA); rabbit anti-glutamine synthetase antibody (Abcam, Cambridge, USA); rabbit anti-Olig2 antibody (EMD Millipore, Burlington, USA); rabbit anti-Iba1 antibody (Wako, Japan); rabbit anti-CamKII antibody (Abcam, Cambridge, USA); mouse anti-GAD67 antibody (EMD Millipore, Burlington, USA); rabbit anti-ChAT antibody (EMD Millipore, Burlington, USA); mouse anti-calbindin antibody (Abcam, Cambridge, USA) and rabbit anti-TH antibody (Novus Biologicals, Littleton, USA). Sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, USA).

To identify the neurons transduced by each vector and to examine the different neuronal subtypes targeted by AAV-F, a Zeiss Axio Imager Z epifluorescence microscope equipped with AxioVision software and a 60× objective was used to obtain high-resolution images illustrating co-localization between GFP and each cellular marker.

Visualization and quantification of overall GFP signal coverage

To quantify the total native GFP fluorescence signal in brain and liver sections, a Virtual Slide Microcope VS120 robotic slide scanner (Olympus) was used to image the entire slide batch in one go using an Olympus UPLSAPO 10× objective. To reduce variability, the entire slide batch was imaged in one round. The initial exposure time for GFP was adjusted so that the fluorescent signal was not oversaturated across the experimental group and remained constant throughout the batch scan. The order of slides was randomized and remained unknown until the end of statistical analysis. Olympus cellSens Standard software was then used to analyze the percentage coverage of GFP in each brain region. The region of interest (ROI) was initially defined using the ROI-polygon tool and the GFP-positive area in this initial ROI was quantified, followed by a similar detection threshold on the GFP channel for all slides analyzed (one threshold was set at the same level for the analysis of all mouse brain sections, and another threshold was used for the analysis of all mouse liver sections and all rat brain sections). The percentage of GFP-positive area was then calculated relative to the total ROI surface area. Autofluorescence signal was taken into account in our analysis, as we set the threshold for eGFP fluorescence intensity above the autofluorescence level (making sure to only include the signal from AAV-GFP-transduced cells). In addition, we imaged each ROI for each brain section, but avoided the outermost regions of the sections, as these regions may also have high levels of autofluorescence. The ventricular space was also excluded from the analysis. Finally, three technical replicates (brain sections) were evaluated per mouse, and all measurements were made blinded until the end of the analysis.

Stereology-based quantitative analysis of the percentage of transduced astrocytes and neurons

Stereology-based studies were performed as previously ^{described24,25} after co-staining brain sections for GFP and NeuN (a neuronal marker) or GFP and GS (glutamine synthetase, a marker of all astrocytes). We did not include microglial cells and oligodendrocytes in this analysis because these cell types were not transduced in significant numbers. Stereological assessment of the percentage of neurons and astrocytes transduced with AAV was performed blinded after identification of the initially injected vector on the moving stage of an Olympus BX51 epifluorescence microscope equipped with a DP70 CCD digital camera, an X-Cite fluorescent lamp, and the corresponding CAST stereology software version 2.3.1.5 (Olympus, Tokyo, Japan). The cerebral cortex was initially outlined under a 4× objective lens. A random subset of the selected area was defined with an optical dissection probe using CAST software. To estimate the percentage of astrocytes or neurons transduced with AAV9, AAV9-PHP.B, AAV-S, and AAV-F, cell counts were performed based on stereological analysis under a 20× objective, with a sampling meander of 10% for the cortical surface in the case of “high transducing” AAVs and 20% for “low transducing” AAVs (to account for the rare GFP-positive cells in these cases). For each subset, the total number of astrocytes (GS-positive cells) or neurons (NeuN-positive cells) was estimated, and the percentage of GFP-positive cells was determined for each of these populations. Only glial cells and neurons with DAPI-positive nuclei within a given subset were counted.

Quantification of vector genome in brain and liver

One hemisphere of the brain and a small piece of liver were frozen for AAV genome extraction for vector genome biodistribution analysis. For fresh frozen brain and liver samples, we isolated genomic and AAV vector DNA from 10 mg of tissue using the DNeasy DNA Isolation Kit from Blood and Tissue (Qiagen) according to the manufacturer's instructions. DNA quantification was performed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). We then performed Taqman qPCR using 50 ng of genomic DNA as a template using a probe and primers specific for the polyA region of the transgene expression cluster (the same assay was performed for titration of purified AAV vectors). To ensure equal input of genomic DNA for each sample, separate qPCR was performed for each sample using a Taqman probe and primer set that detects GAPDH genomic DNA (Thermo Fisher Scientific, Assay ID Mm01180221_g1; gene symbol Gm12070). For each organ/tissue, the copy number of AAV vector genomes was adjusted for each sample to account for any differences in GAPDH Ct values using the following formula: (copy number of AAV vector genomes)/(2 ^ΔCt ). The ΔCt value was calculated using the following formula: GAPDH Ct value for the sample of interest - the average GAPDH Ct value for the sample that had the lowest amount of GAPDH (highest Ct value). Data were expressed as AAV vector genomes per 50 ng of genomic DNA.

Transduction of human neurons :

Primary human fetal neural stem cells (NSCs) were obtained from the Birth Defects Research Laboratory (University of Washington, Seattle, WA) under NIH ethical guidelines. The isolation procedure was described previously ²⁶ with minor modifications. Briefly, brain tissue was incubated in 0.25% trypsin, DNase (90 units/mL) buffer diluted in Hanks balanced salt solution (HBSS) for 45 min. Tissue was titrated, transferred to heat-inactivated fetal bovine serum at 4°C, and centrifuged at 500× g for 20 min. The pellet was resuspended in complete NSC medium consisting of x-Vivo 15 reagent (phenol red and gentamicin free; Lonza) supplemented with 10 μg basic fibroblast growth factor (Life Technologies), 100 μg epidermal growth factor (Life Technologies), 5 μg leukemia inhibitory factor (EMD Millipore), 60 ng/ml N- acetylcysteine (Sigma-Aldrich), 4 ml neural cell survival factor-1 supplement (Lonza), 5 ml 100× N-2 supplement (Life Technologies), 100 units penicillin, 100 μg/ml streptomycin (Life Technologies), and 2.5 μg/ml fungizone (Life Technologies). The supernatants were then filtered through a 40 μm cell strainer (Corning Life Science). Neurospheres >40 μm in diameter were dissociated using Accutase (10 min). Neuronal differentiation medium consisted of 1× Neurobasal medium, 2% serum-free B-27 supplement, and 2 mM GlutaMAX-I supplement (all from Invitrogen) supplemented with human recombinant brain-derived neurotrophic factor (BDNF) (10 ng/ml; Peprotech).

Differentiating NSCs were cultured on glass slides in differentiation medium chamber for 2 weeks and then treated with the indicated AAV vector encoding GFP (7× ¹⁰⁹ VG/well, 150 VG/cell were added). One week after transduction, cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100 (Sigma-Aldrich) in 1× phosphate-buffered saline (PBS; Invitrogen). Cells were stained with the primary monoclonal antibody (TU-20) against neuron-specific class III β-tubulin (1:50; Abcam). Secondary antibodies conjugated to Alexa Fluor 594 (1:200 dilution; Invitrogen) were added for 1 h, followed by DAPI for 30 min. Slides were then treated with ProLong destaining reagent (Invitrogen). Maximum projection images were obtained from captured Z-stacks on a Nikon A1R confocal microscope.

Z-stacks were loaded into Imaris and the surface module was then used to transform the images into a 3D volume. GFP ⁺ neurons (in green channel 1) and class III β-tubulin positive neurons (in red channel 2) were then counted. Using the Imaris colocalization module, a population of neurons doubly positive for the above components was identified.

Statistical analysis

Statistical analysis of the data was performed using GraphPad Prism software (version 8.00). For the different AAV9, AAV9-PHP.B, AAV-S, and AAV-F groups, one-way ANOVA followed by Tukey's multiple comparison test was performed. A p value of <0.05 was considered statistically significant. Results are presented as mean±SEM. Similar analyses were performed for AAV genome biodistribution in the brain and liver, and for human neuronal transduction.

Direct stereotactic injection of vectors

Adult C57BL/6 mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg/kg and 50 mg/kg body weight, respectively) and placed on a stereotaxic frame (Kopf Instruments, Tujunga, USA). Vectors were injected into the cerebral cortex (somatosensory cortex) and hippocampus. A total of 3 μl of viral suspension (1.65× ¹⁰¹⁰ and 5.6× ¹⁰¹⁰ GC per injection site for AAV-F and AAV-S, respectively, at a rate of 0.15 μl/min) was injected using a sharp 33-gauge needle of a 10 μl Hamilton syringe (Sigma-Aldrich, St. Louis, USA). Stereotaxic coordinates of injection sites were calculated using bregma (coordinates of the cerebral cortex: anteroposterior region -1 mm, mediolateral region ±1 mm and dorsoventral region -0.8 mm; coordinates of the hippocampus: anteroposterior region -2 mm, mediolateral region ±1.7 mm and dorsoventral region -2.5 mm).

Intrathecal administration of a loading dose (IT loading dose)

Adult C57BL/6 mice were anesthetized with isoflurane. After the lumbar skin was shaved and cleaned, a 3-4 cm midsagittal incision was made through the skin, exposing the muscles and spine. A catheter was inserted between the L4-L5 region of the spine and connected to a gas-tight Hamilton syringe using a 33-gauge steel needle. Ten microliters of AAV9-CBA-GFP (1.25 × 10 ¹¹ VG) or AAV-F-CBA-GFP (8.8 × 10 ¹⁰ VG) vectors were slowly injected at 2 μl/min. Mice were sacrificed three weeks after injection.

To obtain low-magnification images of all spinal cord and brain sections, overnight staining with anti-GFP antibody (Invitrogen, catalog #G10362, dilution 1:250) was performed, followed by secondary antibody staining and visualization as shown in Figure 3.

For immunostaining of different brain and spinal cord cell types, fixed tissue sections were washed with PBS, blocked with goat serum, and permeabilized with 0.3% Triton in PBS. Target antibodies were diluted in blocking buffer and incubated at 4°C overnight.

First antibodies:

anti-GFP antibody: catalog number ab1218 (abcam): dilution 1:1000

anti-GFAP antibody: catalog number catz0334 (Dako); dilution 1:500

anti-NeuN antibody: catalog number ab177487 (abcam); dilution 1:300

After overnight incubation, slides were washed thoroughly with PBS containing 0.1% Tween. Fluorochrome-conjugated secondary antibodies were then added (1:500 dilution) and incubated for 1 hour at room temperature. After washing, slides were rinsed with PBS and mounted using DAPI mounting solution (ThermoFisher, catalog # P36931). Slides were imaged on a Zeiss LSM 800 confocal laser scanning microscope.

Transmission electron microscopy

Carbon-coated grids (Electron Microscopy Sciences, EMS) were rendered hydrophilic by exposing them to a 25 mA glow discharge for 20 sec. Previously, 5 μl of each AAV vector was adsorbed onto the grid for 1 min and stained with 1% uranyl acetate (EMS #22400) for 20 sec. Grids were evaluated in a TecnaiG ² Spirit BioTWIN and imaged with an AMT 2k charge-coupled device (CCD) camera. Studies were performed in the Electron Microscopy Laboratory of Harvard Medical School. Counting was performed as follows: 5 representative images of each vector preparation were taken; then all full and empty capsids were counted in a counter in Photoshop (CS6). The percentage of empty capsids was calculated for each image and the data were plotted.

Example 1: Construction of an expression-based AAV iTransduce library

We first constructed an AAV library plasmid that consisted of an ITR-flanked AAV2 expression cassette composed of the chicken beta-actin (CBA)-derived Cre recombinase and the AAV9 capsid under the control of the p41 promoter (schematically shown in Fig. 1a) . Pseudorandomized 21-base sequences were inserted between the AAV9 VP1 nucleotides encoding amino acids 588/589 by PCR. Prior to virus packaging, this plasmid library was sequenced by low-level next-generation sequencing (NGS) and confirmed to contain 21-mer inserts in the vast majority of plasmids and to be free of variant bias (data not shown). The capsid library was then packaged and NGS was performed to ensure that the vector generation process retained sufficient diversity to permit selection. The iTransduce library is based on each unique capsid carrying both its own cap gene and a Cre expression construct ( Fig . 1b) . Transgenic mice (Ai9) carrying the floxed-STOP tdTomato cluster were injected intravenously with the AAV library ( Fig. 1b-i) . Capsids that successfully transduce cells enable tdTomato expression in any target organ or cell type (regardless of the availability of specific Cre transgenic mouse lines); and these tdTomato-positive cells can then be sorted by flow cytometry from the tissue of interest (optionally together with cell-specific markers, Fig. 1b-ii) . Viral DNA isolated from these cells must correspond to capsid variants that can efficiently overcome all extracellular and intracellular biological barriers to transgene expression ( Fig. 1b-iii) .

Example 2. Screening of novel AAV9 capsid variants based on transgene expression to identify functional capsids.

To test the iTransduce library strategy, we performed a proof-of-concept screen to isolate AAV capsid variants capable of transducing brain cells following systemic injection. 1.27× ¹⁰¹¹ vector genomes (5× ¹⁰¹² VG/kg) from the library were injected intravenously into the tail vein of one adult male and one female Ai9 mouse, and three weeks after injection, the mice were sacrificed; liver, brain, spleen, and kidney sections were then collected and immunostained for tdTomato. We readily detected tdTomato-positive cells (likely hepatocytes) in the liver, with scattered tdTomato-positive cells (both neurons and astrocytes) in the brain as well as other organs ( Fig. 7a) . We also tested whether the 21-mer insert-containing sap DNA could be preserved by PCR and detected specific bands in 10 organs/tissues from library-injected mice but not from uninjected control mice ( Fig. 7b ) . The remaining brain tissue from male and female mice was then pooled and DNA was extracted, first from whole brain tissue for the first round of selection. The 21-mer insert-containing sap DNA was then amplified and analyzed for identity and read count by NGS. A significant enrichment of the specific peptides in the library population collected from brain tissue after one round of selection was then found compared to the unselected library and to variants isolated from liver ( Fig. 7c , data not shown). The region of viral DNA containing the insert was then isolated and this region was re-cloned back into the AAV plasmid backbone, followed by re-packaging of the capsids (the "brain capsid-enriched library") for a second round of selection.

For the second round of selection, two Ai9 mice (one male and one female) were injected with the library obtained in the first round (1.91× ¹⁰¹⁰ VG, 7.64× ¹⁰¹¹ VG/kg) and the mice were sacrificed three weeks later. Before injection, the sequence containing the modified region was amplified and sequenced by NGS to ensure that no pre-existing bias was introduced into the vector pool ( Fig. 2) . Brain tissue was dissociated to obtain a cell suspension for flow cytometric sorting of tdTomato-positive cells. We cell sorted 3834 tdTomato-positive cells (0.043% of the original cell suspension) by flow cytometry and confirmed successful transduction ( Fig. 8a–b) . Viral DNA from sorted tdTomato cells was amplified and sequenced as previously ( Fig. 2 ) . Viral DNA isolated from tdTomato-positive cells contained 97% of the reads represented by just three peptides, STTLYSP, FVVGQSY, and FQPCP* (where * denotes a stop codon) ( Fig. 2b ) . We selected two of these, STTLYSP (named AAV-S) and FVVGQSY (named AAV-F), for in vivo functional evaluation (we did not evaluate FQPCP* as it was likely a cross-folding product). As seen in Fig. 2 , both of these sequences were detected in the second round library at low levels (~0.4% of reads for each variant), but were highly enriched in the brain after selection.

Example 3. AAV-F capsid mediates efficient transgene expression in the mouse CNS.

To test whether these peptides expressed on the AAV capsid could mediate efficient transgene expression by the AAV vector, we generated capsids (AAV-S and AAV-F) packaging a single-chain GFP expression cassette under the control of the CBA promoter ( Fig. 3a) . For comparison, the parental AAV9 vector and AAV9-PHP.B, the most widely studied AAV9 variant with a 7-mer peptide insert (TLAVPFK) generated by directed evolution, were included ¹² . All vectors were produced at fairly high levels and showed slightly lower production efficiencies than AAV9 ( Table 4).

Table 4. Efficiency of AAV capsid production*

AAV capsid Titer (VG/ml) Production efficiency (VG/cell) AAV9 3.20×10 ¹³ (±2.55×10 ¹³ ) 3.06×10 ⁴ (± 1.39×10 ⁴ ) AAV-F 5.51×10 ¹² (±4.23×10 ¹² ) 9.57×10 ³ (± 8.00×10 ³ ) AAV-S 1.88×10 ¹³ (1.38×10 ¹³ ) 2.09×10 ⁴ (± 5.65×10 ³ )

* All capsids are packaged in a single-stranded cluster with the AAV-CBA-GFP-WPRE transgene flanked by the AAV2 ITR.

Adult male C57BL/6J mice were injected into the lateral tail vein with either a low dose or a high dose (1× ¹⁰¹¹ VG and 8× ¹⁰¹¹ VG vector, respectively, approximately 4× ¹⁰¹² and 3.2× ¹⁰¹³ VG/kg) of one of the following vectors: AAV9, AAV9-PHP.B, AAV-S, and AAV-F (n=3 each). Three weeks after injection, mice were sacrificed and organs were collected for endogenous (unstained) fluorescence analysis of GFP. We quantified the percentage of GFP signal coverage in serial sagittal brain sections (3 sections were analyzed per animal). Remarkably, AAV-F demonstrated a 119-fold (p < 0.0001) and 68-fold (p = 0.0004) increase in GFP fluorescence coverage compared to the parental AAV9 vector at 1 × 10 ¹¹ VH and 8 × 10 ¹¹ VH, respectively ( Fig. 3b, c, e, f; Fig. 9A–B) . AAV9 and AAV-S displayed similar levels of GFP coverage ( Fig. 3b, c, e, f) . AAV9-PHP.B yielded slightly higher GFP coverage at the low dose compared to AAV-F, while similar levels of GFP coverage were observed at the 8 × 10 ¹¹ VH dose ( Fig. 3b, c, e, f). Similar to AAV9-PHP.B, AAV-F penetrated the spinal cord with superior efficiency ( Fig. 3d) . Most brain regions were effective targets for AAV-F, and robust GFP signal was observed in the cerebral cortex, hippocampus, striatum, cerebellum, and olfactory bulb ( Fig. 3f) .

To obtain detailed information on the cell types targeted by AAV-S and AAV-F, we then performed a series of immunostains for GFP and markers of neurons (NeuN), astrocytes (glutamine synthetase, GS), microglial cells (Iba-1) and oligodendrocytes (Olig2). AAV-F and AAV-S, similar to the other two reference vectors, mainly transduced neurons and astrocytes (neither variant appeared to efficiently transduce microglial or oligodendroglial cells, Fig. 4a, b) . Stereological quantification of neurons and astrocytes in the cerebral cortex at a dose of 1× ¹⁰¹¹ VG confirmed the efficient transduction potential of AAV-F compared to standard AAV9 by 65-fold in astrocytes and 171-fold in neurons, while the difference between AAVS and AAV9 was not significant (the percentage of GFP-positive astrocytes was 0.63%±0.24% for AAV9 and 0.36±0.15% for AAV-S, respectively, and the percentage of GFP-positive neurons was 0.039%±0.02% for AAV9 and 0.029±0.002% for AAV-S; all ± before numbers indicate standard error of the mean, SEM). It is noteworthy that AAV-F targets astrocytes to a significant extent (40.78±0.73%) compared to AAV9-PHP.B (28.21±0.25%), and the opposite is true for neurons (6.67±0.5% for AAV-F and 10.59±0.16% for AAV9-PHP.B, Fig. 4c), suggesting a slightly different tropism between these two vectors in mice. Furthermore, AAV-F transduced neurons of various subtypes, including excitatory (CamKII-positive) and inhibitory (GAD67-positive) neurons in the cerebral cortex, dopaminergic neurons in the striatum (expressing tyrosine hydroxylase, TH), Purkinje neurons in the cerebellum (calbindin-positive), and motor neurons in the spinal cord (expressing the choline acetyltransferase marker, ChAT, Fig. 10a). Consistent with the stereological indices of the cerebral cortex ( Fig. 4c) and the high dose AAV-F images compared to AAV9 ( Fig. 3f) , we found efficient transduction of neurons and astrocytes by AAV-F but not AAV9 at a dose of 1× ¹⁰¹¹ Vg/mouse in the striatum, hippocampus and cerebellum ( Fig. 10b) .

To better understand whether the high levels of GFP transgene expression in the brain by AAV-F correspond to higher levels of AAV genomes in the brain, we isolated vector and mouse genomic DNA from liver and brain and performed qPCR at a dose of 8 × 10 ¹¹ Vg/mouse. AAV-F showed a 20-fold increase (p < 0.0001) in AAV genomes in the brain compared to AAV9 ( Fig. 4d) . As reported, AAV9-PHP.B had a much higher (25-fold) number of AAV genomes in the brain compared to AAV9, while AAV-S had a low level in accordance with the GFP fluorescence data ( Fig. 3) . PHP.B showed expression levels in the liver that were slightly lower than those of AAV9 and AAV-F (but not AAV-S; Fig. 4d) . AAV-F levels in the liver were similar to those of AAV9, and AAV-S showed a lower but non-significant downward trend. We also assessed the biodistribution of AAV-F compared to AAV9 in several other organs, namely skeletal muscle, heart and spinal cord ( Fig. 11) . No significant difference was observed between the two vectors in muscle or heart (although a trend towards higher AAV-F expression was observed in the heart). As expected from the expression in the brain, AAV-F showed significantly higher expression levels in the spinal cord compared to AAV9 (p < 0.05, t-test).

To investigate transgene expression in peripheral organs following systemic injection, we analyzed GFP fluorescence in liver, heart, skeletal muscle, and retina ( Fig. 4e). Not surprisingly, all vectors efficiently penetrated the liver. In heart and skeletal muscle, AAV-S yielded higher amounts of bright GFP signals/section than AAV9 and AAV-F. All capsids mediated low transduction in the retina, as expected from intravenous delivery. Interestingly, although AAV-9 and AAV-S did not penetrate the neuronal retina, corresponding expression was observed in the retinal pigment epithelium (RPE). Both AAV9-PHP.B and AAV-F penetrated multiple retinal layers, primarily the ganglion cell layer (GCL). GFP expression was also observed in the inner nuclear layer (INL), with significant expression in the outer plexiform layer (OPL), which may indicate bipolar or inhibitory horizontal cell transduction.

Because transduction by AAV vectors in mice can vary significantly depending on the sex and strain of mice, we investigated whether the most effective capsid, AAV-F, could efficiently enter the brain after systemic injection into C57BL/6 females as well as BALB/c males. Mice were injected with 1×10¹¹VG (4×10¹²VG/kg), resulting in robust and efficient transduction independent of strain or sex (Fig. 5a-c). These results were in contrast to the lack of efficacy of AAV9-PHP.B in central nervous system transduction following systemic delivery to BALB/c mice, as reported previously (fig.5b, c) ^13,14 .

Although iodixanol density gradient purification removes most of the empty capsids, to test whether the excess of empty capsids in AAV-F could explain its increased brain biodistribution compared to AAV9, we performed transmission electron microscopy (TEM) on two separate AAV-F preparations and compared them with two independent AAV9 preparations. As shown in Fig. 12A–E , no significant difference in empty capsid levels was observed between AAV-F and AAV9 (mean 4.63±1.99% vs. 5.2±2.18% empty capsids for AAV9 and AAV-F, respectively, mean±SD, p=0.54, unpaired t-test).

We next analyzed whether AAV-F could be used as a vector for transduction into the CNS by other routes of administration. We first tested AAV-S and AAV-F for mediating transgene expression in the brain following direct injection into the hippocampus of adult C57BL/6 mice. We found that both capsids achieved broad GFP expression following direct injection, primarily in neurons ( Fig. 13) . Intrathecal injection of AAV vectors for transduction into the spinal cord has shown promise for treatment of this organ. One drawback is the limited vector distribution into the brain following vector injection into the lumbar region. We compared AAV9 and AAV-F following intrathecal bolus injection of vector into the lumbar region of the spinal cord in adult C57BL/6 mice. Three weeks after injection, the mice were sacrificed and the spinal cord and brain were analyzed. Notably, AAV-F resulted in much more intense GFP expression throughout the spinal cord compared to AAV9, and transduction was observed in both white and gray matter. Meanwhile, AAV9 transduction was restricted primarily to white matter. Strikingly, astrocyte and neuronal transduction was also detected in the brains of AAV-F-injected mice, but not AAV9 ( Fig. 14) .

Example 4. AAV-F mediates enhanced transduction of human neurons.

Since the selection was performed in mice, an analysis was performed to determine whether the robust transduction properties of AAV-F were translated to human cells. Primary neurons derived from human stem cells were transduced with equal doses of AAV9, AAV-S, and AAV-F, all of which encoded GFP. One week later, they were fixed, stained with class III β-tubulin, and analyzed for the percentage of GFP-positive neurons. Notably, AAV-F transduced 62% of neurons, a three-fold increase over AAV9 (p < 0.05) ( Fig. 6a, b) . AAV-S produced a small but statistically significant (p < 0.05) increase in transduction efficiency compared to AAV9 ( Fig. 6a) .

Example 5: AAV-S penetrates the inner ear with high efficiency

In recent years, new AAV vectors have been developed that are designed to target specific organ and cell types with high efficiency. Many of these use 7-mer peptide inserts that alter the transduction properties of a specific AAV serotype. However, these vectors can often be used to transduce other tissues, including difficult-to-treat organs such as the inner ear. Transduction of specific inner ear cell types, such as hair cells, remains a challenge, as many traditional AAV vectors cannot adequately target one class of hair cells, namely outer hair cells (OHCs).

As noted above, one vector (AAV-F) showed great promise and high expression in the CNS after systemic injection, whereas the other (AAV-S) did not. Although systemically administered AAV-S cannot efficiently cross the blood-brain barrier, it does penetrate into various tissues including the heart, liver, and muscle. Following direct injection into the brain, AAV-S showed high local transduction efficiency in neurons even at relatively low doses. To test its transduction properties in the inner ear, we generated AAV-S encoding a single-stranded expression cassette initiating EGFP production under the control of the CBA promoter and injected this vector into neonatal (P1) mice through the round window. We found that AAV-S penetrated cochlear hair cells very well. Both inner and outer hair cells were transduced with up to 100% and 99% efficiencies ( Fig. 15a, b) at the tested dose (2×10 ¹⁰ VG). We also observed significant transduction of the spiral limbus and spiral ganglia ( Fig. 15c, d) . Overall, as shown in this application, AAV-S can be used for gene therapy of the inner ear.

Example 6: Use of the iTransduce system to screen non-transgenic adult primates for capsids that efficiently penetrate fibroblasts.

2-3 rounds of selection for fibrocyte-targeted AAV capsids were performed in non-human primates such as cynomolgus monkeys. To identify fibrocyte selectivity, selection for a transduction-competent AAV capsid was performed by co-injecting an iTransduce AAV library with AAV9-PHP.B, which encodes a GJB2 -floxed-STOP-tdTomato cluster (size matched to AAV capsid size). In the NHP inner ear, AAV9-PHP.B-CBA-GFP targeted multiple cochlear cells including fibrocytes, HC, and the spiral ganglia neuronal region. In this selection strategy (see Fig. 16A), tdTomato expression was restricted to fibrocytes under the control of the GJB2 promoter, essentially creating a transgenic NHP inner ear. AAV capsids that enter fibrocytes and include the tdTomato gene are selected by flow cytometry from dissociated snails, and capsid DNA preserved for NGS is cloned to identify peptide sequences that mediate AAV-mediated expression in fibrocytes. After enrichment for individual peptides, deep sequencing is performed as described above to provide information on when to stop additional rounds of selection (likely when a particular peptide represents >25% of reads).

Alternatively or additionally, 2-3 rounds of selection for AAV capsids targeting spinal cord cells were performed in non-human primates such as cynomolgus monkeys. To identify spinal cord selectivity, selection for transduction-competent AAV capsids was performed by co-injection of the iTransduce AAV library with AAV9, which encodes a CBA -floxed-STOP-mPlum cluster (size matched to AAV capsid size). In the NHP spinal cord, AAV9 enters cells including neurons and astrocytes. In this selection strategy (see Fig. 16B), AAV capsids that enter spinal cord cells and trigger mPlum fluorescence are selected by flow cytometry from dissociated spinal cord, and capsid DNA saved for NGS is cloned to identify peptide sequences that mediate AAV-mediated expression in spinal cord cells. After enrichment for individual peptides, deep sequencing is performed as described above to provide information on when to stop additional rounds of selection (likely when a particular peptide represents >25% of the reads).

Following identification of candidate capsid clones, capsids were converted as previously described into vectors encoding the GFP cluster. Capsids were then tested for transduction of target cells following direct injection through the round window membrane (RMW) (e.g., as shown in Fig. 16A) or intrathecal injection (e.g., as shown in Fig. 16B).

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Other embodiments of the invention

It should be noted that although the present invention has been described in detail above, such description is provided for illustrative purposes only and is not considered as limiting the scope of the invention, which is defined by the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

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LIST OF SEQUENCES

<110> THE GENERAL HOSPITAL CORPORATION

PRESIDENT AND FELLOWS OF HARVARD COLLEGE

<120> ENGINEERED ADENO-ASSOCIATED (AAV) VECTORS

FOR TRANSGENE EXPRESSION

<130> 29539-0390WO1

<140> PCT/US2020/025720

<141> 2020-03-30

<150> 62/825,703

<151> 2019-03-28

<160> 149

<170> PatentIn version 3.5

<210> 1

<211> 7

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<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 1

Ser Thr Thr Leu Tyr Ser Pro

1 5

<210> 2

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 2

Phe Val Val Gly Gln Ser Tyr

1 5

<210> 3

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 3

Phe Gln Pro Cys Pro

1 5

<210> 4

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

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<400> 4

Pro Ser Tyr Leu Thr Thr Ser

1 5

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 5

Tyr Ser Gln Gly Val Val Phe

1 5

<210> 6

<211> 736

<212> PRT

<213> Adeno-associated virus 9

<400> 6

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 7

<211> 743

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 7

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Phe Val Val Gly

580 585 590

Gln Ser Tyr Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile

595 600 605

Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro

610 615 620

Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro

625 630 635 640

Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile

645 650 655

Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp

660 665 670

Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val

675 680 685

Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro

690 695 700

Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe

705 710 715 720

Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr

725 730 735

Arg Tyr Leu Thr Arg Asn Leu

740

<210> 8

<211> 2229

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polynucleotide

<400> 8

atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60

gagtggtggg ctttgaaacc tggagccct caacccaagg caaatcaaca acatcaagac 120

aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840

tggggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140

acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200

ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260

cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320

gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380

ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440

ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500

tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560

ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620

ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680

accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740

gccacaaacc accaagtgc ccaatttgtt gttggtcaga gttatgcaca ggcgcagacc 1800

ggctgggttc aaaaccaagg aatacttccg ggtatggttt ggcaggacag agatgtgtac 1860

ctgcaaggac ccatttgggc caaaattcct cacacggacg gcaactttca cccttctccg 1920

ctgatgggag ggtttggaat gaagcacccg cctcctcaga tcctcatcaa aaacacacct 1980

gtacctgcgg atcctccaac ggccttcaac aaggacaagc tgaactcttt catcaccag 2040

tattctactg gccaagtcag cgtggagatc gagtgggagc tgcagaagga aaacagcaag 2100

cgctggaacc cggagatcca gtacacttcc aactattaca agtctaataa tgttgaattt 2160

gctgttaata ctgaaggtgt atatagtgaa ccccgcccca ttggcaccag atacctgact 2220

cgtaatctg 2229

<210> 9

<211> 743

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 9

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ser Thr Thr Leu

580 585 590

Tyr Ser Pro Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile

595 600 605

Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro

610 615 620

Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro

625 630 635 640

Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile

645 650 655

Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp

660 665 670

Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val

675 680 685

Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro

690 695 700

Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe

705 710 715 720

Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr

725 730 735

Arg Tyr Leu Thr Arg Asn Leu

740

<210> 10

<211> 2229

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polynucleotide

<400> 10

atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60

gagtggtggg ctttgaaacc tggagccct caacccaagg caaatcaaca acatcaagac 120

aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840

tggggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140

acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200

ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260

cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320

gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380

ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440

ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500

tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560

ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620

ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680

accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740

gccacaaacc accaagtgc ccaatctact acgctttata gtcctgcaca ggcgcagacc 1800

ggctgggttc aaaaccaagg aatacttccg ggtatggttt ggcaggacag agatgtgtac 1860

ctgcaaggac ccatttgggc caaaattcct cacacggacg gcaactttca cccttctccg 1920

ctgatgggag ggtttggaat gaagcacccg cctcctcaga tcctcatcaa aaacacacct 1980

gtacctgcgg atcctccaac ggccttcaac aaggacaagc tgaactcttt catcaccag 2040

tattctactg gccaagtcag cgtggagatc gagtgggagc tgcagaagga aaacagcaag 2100

cgctggaacc cggagatcca gtacacttcc aactattaca agtctaataa tgttgaattt 2160

gctgttaata ctgaaggtgt atatagtgaa ccccgcccca ttggcaccag atacctgact 2220

cgtaatctg 2229

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

primer

<400> 11

gtactatctc tctagaacta ttaacggttc 30

<210> 12

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

primer

<220>

<221> modified_base

<222> (41)..(41)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (43)..(44)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (46)..(47)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (49)..(50)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (52)..(53)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (55)..(56)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (58)..(59)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (61)..(62)

<223> a, c, t, g, unknown or other

<400> 12

gtattccttg gttttgaacc caaccggtct gcgcctgtgc nmnnmnnmnn mnnmnnmnnm 60

nnttgggcac tctggtggtt tgtg 84

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

primer

<400> 13

aatcctggac ctgctatggc 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

primer

<400> 14

tgccaaacca tacccggaag 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

primer

<400> 15

agctaccgac aacaacgtgt 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

primer

<400> 16

agaagggtga aagttgccgt 20

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 17

cagcctcgtc ttactgagct t 21

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 18

Gln Pro Arg Leu Thr Glu Leu

1 5

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 19

agtcatttta gtaattctat g 21

<210> 20

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 20

Ser His Phe Ser Asn Ser Met

1 5

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 21

ccgccttcta cggggaatcc t 21

<210> 22

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 22

Pro Pro Ser Thr Gly Asn Pro

1 5

<210> 23

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 23

aatagtcttg gggtgatgga t 21

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 24

Asn Ser Leu Gly Val Met Asp

1 5

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 25

cctcctatgg ctacgcctca g 21

<210> 26

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 26

Pro Pro Met Ala Thr Pro Gln

1 5

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 27

ccgcatactc gggctgagcc t 21

<210> 28

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 28

Pro His Thr Arg Ala Glu Pro

1 5

<210> 29

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 29

gtgaatcaga ctaattattc t 21

<210> 30

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 30

Val Asn Gln Thr Asn Tyr Ser

1 5

<210> 31

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 31

catgcgccga ggctggttca g 21

<210> 32

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 32

His Ala Pro Arg Leu Val Gln

1 5

<210> 33

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 33

tggagtaagg agtcgaatca t 21

<210> 34

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 34

Trp Ser Lys Glu Ser Asn His

1 5

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 35

actactgctg gtccgactga g 21

<210> 36

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 36

Thr Thr Ala Gly Pro Thr Glu

1 5

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 37

tcgtggccgc agtcgccttc g 21

<210> 38

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 38

Ser Trp Pro Gln Ser Pro Ser

1 5

<210> 39

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 39

acttgtgctg ttccggatcg g 21

<210> 40

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 40

Thr Cys Ala Val Pro Asp Arg

1 5

<210> 41

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 41

cctcgtgcgg agcctcggac g 21

<210> 42

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 42

Pro Arg Ala Glu Pro Arg Thr

1 5

<210> 43

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 43

cctactgcgg gtgtgtatct g 21

<210> 44

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 44

Pro Thr Ala Gly Val Tyr Leu

1 5

<210> 45

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 45

cagttgagta atacgacgtt g 21

<210> 46

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 46

Gln Leu Ser Asn Thr Thr Leu

1 5

<210> 47

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 47

cggatggata tggctgtgag g 21

<210> 48

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 48

Arg Met Asp Met Ala Val Arg

1 5

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 49

cggccgccgc ctgcgactcc t 21

<210> 50

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 50

Arg Pro Pro Pro Ala Thr Pro

1 5

<210> 51

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 51

cagtatctgtttcatattttt 21

<210> 52

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 52

Gln Tyr Leu Phe His Ile Phe

1 5

<210> 53

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 53

ggtgctggga ctcttacttc t 21

<210> 54

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 54

Gly Ala Gly Thr Leu Thr Ser

1 5

<210> 55

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 55

cctctttcgc gtgttgcttt g 21

<210> 56

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 56

Pro Leu Ser Arg Val Ala Leu

1 5

<210> 57

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 57

actactgcta agacgactgg g 21

<210> 58

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 58

Thr Thr Ala Lys Thr Thr Gly

1 5

<210> 59

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 59

tcgctgccgg ctactcatgt g 21

<210> 60

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 60

Ser Leu Pro Ala Thr His Val

1 5

<210> 61

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 61

agtgagaagg tgattcggag t 21

<210> 62

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 62

Ser Glu Lys Val Ile Arg Ser

1 5

<210> 63

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 63

aagtcgacta tgcggaagcc g 21

<210> 64

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 64

Lys Ser Thr Met Arg Lys Pro

1 5

<210> 65

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 65

gtgtcgaagc agaatacgac t 21

<210> 66

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 66

Val Ser Lys Gln Asn Thr Thr

1 5

<210> 67

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 67

gctgatatga cggggcctac t 21

<210> 68

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 68

Ala Asp Met Thr Gly Pro Thr

1 5

<210> 69

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 69

cctactcgtg cgactcctat t 21

<210> 70

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 70

Pro Thr Arg Ala Thr Pro Ile

1 5

<210> 71

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 71

tcttggcctg ggctgttgct g 21

<210> 72

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 72

Ser Trp Pro Gly Leu Leu Leu

1 5

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 73

tggaggctgg agcagttgaa g 21

<210> 74

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 74

Trp Arg Leu Glu Gln Leu Lys

1 5

<210> 75

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 75

cttccgagtt ctccggtttt t 21

<210> 76

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 76

Leu Pro Ser Ser Pro Val Phe

1 5

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 77

cctcagcggc atcagaatga t 21

<210> 78

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 78

Pro Gln Arg His Gln Asn Asp

1 5

<210> 79

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 79

tcgattccgt ttcggtatcc g 21

<210> 80

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 80

Ser Ile Pro Phe Arg Tyr Pro

1 5

<210> 81

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 81

cctgatggtc cgcaggctct g 21

<210> 82

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 82

Pro Asp Gly Pro Gln Ala Leu

1 5

<210> 83

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 83

tcgactacgt ggaagtctga t 21

<210> 84

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 84

Ser Thr Thr Trp Lys Ser Asp

1 5

<210> 85

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 85

ttgcagaaga tggctgttcc g 21

<210> 86

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 86

Leu Gln Lys Met Ala Val Pro

1 5

<210> 87

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 87

acggggtgta agtgttcgca g 21

<210> 88

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 88

Thr Gly Cys Lys Cys Ser Gln

1 5

<210> 89

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 89

catctgcctg ttatgaatga g 21

<210> 90

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 90

His Leu Pro Val Met Asn Glu

1 5

<210> 91

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 91

gtttcgttga attatactgc t 21

<210> 92

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 92

Val Ser Leu Asn Tyr Thr Ala

1 5

<210> 93

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 93

cctcctcctc ctccgcctgt g 21

<210> 94

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 94

Pro Pro Pro Pro Pro Pro Val

1 5

<210> 95

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 95

agttctactg ctagtcgtcg t 21

<210> 96

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 96

Ser Ser Thr Ala Ser Arg Arg

1 5

<210> 97

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 97

ctgcttgtga cgtgtccgga t 21

<210> 98

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 98

Leu Leu Val Thr Cys Pro Asp

1 5

<210> 99

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 99

tcgttgctgc gttatgctcc g 21

<210> 100

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 100

Ser Leu Leu Arg Tyr Ala Pro

1 5

<210> 101

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 101

cagccgctgc gtccgaatct g 21

<210> 102

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 102

Gln Pro Leu Arg Pro Asn Leu

1 5

<210> 103

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 103

gctttggata ctcatcattg g 21

<210> 104

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 104

Ala Leu Asp Thr His His Trp

1 5

<210> 105

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 105

cggcctactc gtctttctcc g 21

<210> 106

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 106

Arg Pro Thr Arg Leu Ser Pro

1 5

<210> 107

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 107

gatgcttggt cggcttctat t 21

<210> 108

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 108

Asp Ala Trp Ser Ala Ser Ile

1 5

<210> 109

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 109

tataagtgtt ggccgaggag t 21

<210> 110

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 110

Tyr Lys Cys Trp Pro Arg Ser

1 5

<210> 111

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 111

aagtatctga ctaatctttc t 21

<210> 112

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 112

Lys Tyr Leu Thr Asn Leu Ser

1 5

<210> 113

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 113

tcgcggcctc cgccgtctct g 21

<210> 114

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 114

Ser Arg Pro Pro Pro Ser Leu

1 5

<210> 115

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 115

taggcgccga ttctttagtt g 21

<210> 116

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 116

atgcctcctt ctcctccggg t 21

<210> 117

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 117

Met Pro Pro Ser Pro Pro Gly

1 5

<210> 118

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 118

ccgtttcctc agatgctttt t 21

<210> 119

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 119

Pro Phe Pro Gln Met Leu Phe

1 5

<210> 120

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 120

cctcaggcgt ctcatgttaa t 21

<210> 121

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 121

Pro Gln Ala Ser His Val Asn

1 5

<210> 122

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 122

gagtattgtt cttagattcc t 21

<210> 123

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 123

Glu Tyr Cys Ser

1

<210> 124

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 124

tcgtagaatt tgcgggtgtt g 21

<210> 125

<211> 1

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 125

Ser

1

<210> 126

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 126

tattatcatt cgctgtcgtc g 21

<210> 127

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 127

Tyr Tyr His Ser Leu Ser Ser

1 5

<210> 128

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 128

aagcattctc agcggcggaa t 21

<210> 129

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 129

Lys His Ser Gln Arg Arg Asn

1 5

<210> 130

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 130

tagactaatg tgtggattaa t 21

<210> 131

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 131

actaagacgc ctgagcatta g 21

<210> 132

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 132

Thr Lys Thr Pro Glu His

1 5

<210> 133

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 133

gggccggagg ggccgcgtct g 21

<210> 134

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 134

Gly Pro Glu Gly Pro Arg Leu

1 5

<210> 135

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 135

tgtacttcta atctgcatgg g 21

<210> 136

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 136

Cys Thr Ser Asn Leu His Gly

1 5

<210> 137

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 137

gttattactc atctgactga t 21

<210> 138

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 138

Val Ile Thr His Leu Thr Asp

1 5

<210> 139

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 139

gattctaatc attttggtcc g 21

<210> 140

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 140

Asp Ser Asn His Phe Gly Pro

1 5

<210> 141

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 141

ccgagtcagc agacttagat g 21

<210> 142

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 142

Pro Ser Gln Gln Thr

1 5

<210> 143

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 143

tttgttgctg attgggagcc t 21

<210> 144

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 144

Phe Val Ala Asp Trp Glu Pro

1 5

<210> 145

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 145

ctgatgcaga ctaatattac g 21

<210> 146

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 146

Leu Met Gln Thr Asn Ile Thr

1 5

<210> 147

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 147

ctggtggttc ctaatcgtga t 21

<210> 148

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 148

Leu Val Val Pro Asn Arg Asp

1 5

<210> 149

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 149

Thr Leu Ala Val Pro Phe Lys

1 5

<---

Claims (22)

1. An AAV capsid protein for use in an AAV vector, wherein the AAV capsid protein comprises an amino acid sequence that comprises at least seven contiguous amino acids of the sequence STTLYSP (SEQ ID NO:1). 2. The AAV capsid protein of claim 1, wherein the AAV is AAV9. 3. The AAV capsid protein according to claim 1 or 2, comprising AAV9 VP1. 4. The AAV capsid protein of claim 3, wherein a sequence that contains at least seven consecutive amino acids of the sequence STTLYSP (SEQ ID NO:1) is inserted into a position corresponding to amino acids 588 and 589 of the sequence SEQ ID NO:6. 5. A nucleic acid encoding the AAV capsid protein of any one of claims 1 to 4. 6. An AAV vector for use in delivering a nucleic acid into a cell, wherein the AAV vector comprises the AAV capsid protein of any one of claims 1 to 4. 7. The AAV vector of claim 6, wherein the AAV does not comprise a wild-type VP1, VP2, or VP3 capsid protein. 8. The AAV vector according to claim 6 or 7, further comprising a transgene. 9. The AAV vector of claim 8, wherein the transgene is a therapeutic transgene. 10. A method for delivering a transgene to a cell, wherein the method comprises contacting the cell with the AAV vector of claim 8 or 9. 11. The method according to claim 10, wherein the cell is a neuron, a cardiomyocyte, a myocyte, an astrocyte, a glial cell, an inner hair cell, an outer hair cell, a supporting cell, an inner ear fibrocyte, a photoreceptor cell, an interneuron, a retinal ganglion cell, or a retinal pigment epithelial cell. 12. The method according to claim 11, wherein the neuron is a dorsal root ganglion neuron or a spiral ganglion neuron. 13. The method according to any one of claims 10-12, wherein the cell is present in a living individual. 14. The method of claim 13, wherein the individual is a mammal. 15. The method of claim 13 or 14, wherein the cell is present in tissue selected from brain tissue, spinal cord tissue, dorsal root ganglia tissue, heart tissue, inner ear tissue, eye tissue, muscle tissue, and combinations thereof. 16. The method according to any one of claims 13 to 15, wherein the individual suffers from Alzheimer's disease; Parkinson's disease; X-linked adrenoleukodystrophy; Canavan disease; Niemann-Pick disease; spinal muscular atrophy; Huntington's disease; connexin-26-associated disease; Usher disease type 3A; Usher disease type 2D; hair cell associated hearing loss; hair cell associated hearing loss (DFNB7/11); inner hair cell associated hearing loss (DFNB9); Usher disease type 1F; Usher disease type 1B; retinitis pigmentosa (RP; non-syndromic); Leber's congenital amaurosis; Leber's hereditary optic neuropathy; Usher syndrome (RP; a syndrome associated with deafness); Duchenne muscular dystrophy; post-allograft vasculopathy; hemophilia A; or hemophilia B. 17. The method of any one of claims 13-16, wherein the cell is in the brain of an individual and the AAV is administered via parenteral delivery, intracerebral delivery or intrathecal delivery. 18. The method of claim 17, wherein intrathecal delivery is accomplished by lumbar injection, intracisternal injection, or intraparenchymal injection. 19. The method according to any one of claims 10-17, wherein the AAV is administered parenterally. 20. The method of claim 19, wherein the AAV is added intravenously, intraarterially, subcutaneously, intraperitoneally, or intramuscularly. 21. The method of any one of claims 13-16, wherein the cell is in the eye of the individual and the AAV is administered by subretinal injection or intravitreal injection. 22. The method of any one of claims 13-16, wherein the cell is in the inner ear of the individual and the AAV is administered to the cochlea by application to or through the round window membrane, through a surgically drilled cochleostomy located adjacent to the round window, through an opening in the bony oval window, or through the semicircular canal.

Images