Methods and materials for developing AAV vectors and promoters based on single-cell transcriptomes

Abstract

Provided herein are high-throughput methods for creating AAV vectors and / or promoter sequences that have high efficiency and / or specificity for a variety of cell types.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application Serial No. 62 / 785,818, filed on December 28, 2018. The disclosure of that prior application is considered part of the disclosure of this application (and is incorporated herein by reference).

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with government support under Grant MH113095 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art 1. Technical Field

[0006] The present invention relates to methods and materials for developing adeno-associated virus (AAV) vectors and promoters based on single cell transcriptomes. For example, an efficient and high-throughput method for creating an effective AAV vector is provided herein.

[0007] 2. Background Information

[0008] Efficient and targeted gene delivery is fundamental to the success of gene therapy and circuit-based tools such as optogenetics. Adequate levels of gene expression in the desired cell type are critical, and ideally, off-target expression should be minimized for precisely targeted therapy, patient safety, and circuit-specific manipulation.

[0009] An attractive vector system for gene therapy is based on genetically engineered and modified adeno-associated viruses (AAV). However, existing screening methods for creating AAV require larger numbers of cells and focus on only a single cell type over multiple rounds of selection. These are also based on DNA screening. Therefore, more efficient and higher throughput methods are needed for creating AAV vectors for gene therapy. Summary of the invention

[0010] Provided herein are high throughput methods for creating AAV vectors and promoter sequences that have high efficiency and / or specificity for a variety of cell types. In some embodiments, first, a plurality of variants (between about 20, or about 50, or about 100, or about 1000, or about 10,000, or about 10,000, or about 1,000,000 and up to about 10) are created. 6 or about 10 7 In some cases, the AAV libraries provided herein can be directly injected into the desired tissue (e.g., intravitreal injection) or systemically injected.

[0011] Injection (or introduction or infection / transfection) into certain tissues in culture, such as retinal organoids, can also be used in lieu of in vivo screening. These libraries are created so that each AAV variant within the library contains a unique "DNA barcode" (i.e., a unique DNA sequence that is part of the AAV genome and indicates the identity of the viral variant), which allows tracking of the AAV capsid or synthetic upstream promoter.

[0012] Secondly, in some embodiments, after injection, AAV vectors compete with each other in vivo (or in cultured tissues), so that stronger AAV vectors or promoters lead to higher DNA barcode expression levels, and more specific AAV vectors or promoters have increased expression levels in one or more cell types compared to all other cell types. Subsequently, a single cell or single nucleus microfluidics method (from, for example, 10X GENOMICS or DOLOMITE BIO) is used to create a cDNA library of a single cell (or nucleus).

[0013] The best vectors are then identified based on the presence and quantity of DNA barcodes from transcriptomes of many different cell types in parallel, based on specificity, expression level, and / or other desired characteristics. Selection can be done at two levels: (a) highly diverse libraries of viral capsids can be screened for highly efficient and specificity-oriented vectors, and (b) enhancer / promoter constructs can be evaluated for their ability to drive expression in specific cell populations.

[0014] In some cases, the performance of the viral capsid can be evaluated based on mRNA transcription levels rather than DNA. RNA can be used as an indicator of viral function because it reflects the ability of the vector to drive the expression of the loaded protein, rather than just entering the cell. And, as described herein, the methods provided herein can involve the use of a variety of cell types, usually but not limited to in vivo, which is an improvement on conventional methods involving large pieces of tissue or one cell type during multiple rounds of selection.

[0015] In some cases, the methods provided herein can be performed in many tissues, including the retina, brain, muscle or other tissues of primates, to maximize the transformation potential of the resulting vector. In some cases, the high-throughput screening methods provided herein can allow identification and characterization of viral variants and promoters with desired properties, including broad tropism and specificity.

[0016] In general, one aspect of the present invention describes a method comprising: (a) creating an AAV mutant or promoter library, wherein each AAV in the library comprises a unique DNA barcode, or each promoter construct comprises a unique DNA barcode, (b) packaging the AAV mutant or promoter into a packaging cell line using a double (for capsid library) or triple (for some capsid or promoter libraries) transfection scheme, (c) delivering the AAV mutant library into one or more tissues of an animal host, or infecting tissues in culture, (d) maintaining the AAV mutant library in vivo or culturing the mutant library in tissues in culture for a period of time suitable for the AAV vectors in the AAV mutant library to compete with each other in the cultured tissues or one or more tissues of the animal host into which the AAV mutant library has been delivered, and (e) using a single cell or single nucleus microfluidics method to create a single cell or single nucleus cDNA library from cells in one or more tissues of the animal host into which the AAV mutant library has been delivered. Step (e) can use single cell microfluidics technology. Step (e) can use single nucleus microfluidics technology. The one or more tissues of the animal host may include nervous tissue. The nervous tissue may include central nervous system tissue. The central nervous system tissue may be brain tissue. The nervous tissue may include peripheral nervous system tissue. The one or more tissues of the animal host may include retinal tissue. The one or more tissues of the animal host may include muscle tissue. The muscle tissue may include striated muscle. The muscle tissue may include cardiac muscle. The muscle tissue may include smooth muscle. The animal host may be a primate. The primate may be an Old World monkey. The Old World monkey may be a rhesus macaque (Macaca mulatta). The primate may be an ape of the Hylobatidae family or the Hominidae family. The primate may be a primate of the genus non-human. The primate may be a primate of the genus non-chimpanzee (genus Pan). Delivery of the AAV mutant library may be by injection into a tissue.

[0017] On the other hand, described herein is a method for obtaining an AAV mutant having the ability to infect a desired cell type in vivo and to maintain in vivo in the cell type for at least one week. The method comprises (or is essentially composed of or is composed of): (a) introducing an AAV mutant library into an animal host comprising the cell type, wherein each AAV in the library comprises a unique DNA barcode, and (b) identifying one or more AAV mutants based on the barcodes of one or more AAV mutants to be present in cells of the cell type, wherein the cell is maintained in the animal host for at least one week after the library is introduced into the animal host. The cell type may be a central nervous system cell type or a peripheral nervous system cell type. The cell type may be a retinal cell type, a striated muscle cell type, a cardiomyocyte cell type, or a smooth muscle cell type. The animal host may be a primate. The primate may be an Old World monkey. The primate may be a rhesus macaque (Macaca mulatta). The primate may be an ape of the Hylobatidae family or the Hominidae family. The primate may be a primate of the non-human genus. The primate may be a primate other than the genus Pan. The library may be introduced into the animal host by injection into a tissue comprising the cell type. The at least one week may be from 1 week to 12 weeks.

[0018] On the other hand, the present invention describes a method for obtaining a promoter sequence from an AAV virus library. The present method includes (or is essentially composed of or is composed of): (a) introducing the library into an animal host containing the cell type, wherein each AAV in the library contains a unique promoter sequence configured to drive the expression of a fluorescent polypeptide, and (b) identifying one or more promoter sequences based on the expression of the fluorescent polypeptide to be present in cells of the cell type, which are maintained in the animal host for at least one week after the library is introduced into the animal host. The cell type can be a central nervous system cell type or a peripheral nervous system cell type. The cell type can be a retinal cell type, a striated muscle cell type, a cardiomyocyte cell type, or a smooth muscle cell type. The animal host can be a primate. The primate can be an Old World monkey. The primate can be a rhesus macaque (Macaca mulatta). The primate can be an ape of the Hylobatidae family or the Hominidae family. The primate can be a primate of the non-human genus. The primate may be a primate other than the genus Pan. The library may be introduced into the animal host by injection into a tissue comprising the cell type. The at least one week may be from 1 week to 12 weeks.

[0019] In another aspect, described herein are isolated nucleic acids comprising (or consisting essentially of or consisting of) nucleic acids encoding AAV rep polypeptides, nucleic acids encoding AAV cap polypeptides, and nucleic acid cassettes, wherein the nucleic acid cassettes comprise a promoter sequence, a nucleic acid encoding a peptide tag, a nucleic acid barcode, and a poly A tail sequence. The nucleic acid encoding the AAV rep polypeptide, the nucleic acid encoding the AAV cap polypeptide, and the nucleic acid cassettes may be located between two inverted terminal repeats. The nucleic acid barcode may be between 20-30 nucleotides in length. The isolated nucleic acid may be a plasmid.

[0020] In another aspect, isolated nucleic acids are described herein that include (or consist essentially of or consist of) nucleic acids and nucleic acid cassettes encoding AAV cap polypeptides, wherein the nucleic acid cassettes include a promoter sequence, a nucleic acid encoding a fluorescent polypeptide, and a poly A tail sequence, wherein the isolated nucleic acid lacks nucleic acid encoding a full-length rep polypeptide. The nucleic acid and nucleic acid cassette encoding the AAV cap polypeptide may be located between two inverted terminal repeats. The isolated nucleic acid may include a nucleic acid encoding a rep polypeptide amino acid sequence that is no more than 25%, no more than 50%, no more than 75%, or no more than 85% of the amino acid sequence of the full-length rep polypeptide.

[0021] Unless otherwise defined, the meaning of all technical and scientific terms used herein is the same as that generally understood by those of ordinary skill in the art to which the invention belongs. Although the present invention can be implemented with methods and materials similar or equivalent to those described herein, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification including definitions shall prevail. In addition, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0022] The accompanying drawings and the following description further illustrate one or more embodiments of the present invention in detail. Other features, objectives and advantages of the present invention can also be clearly seen from the description, drawings and claims herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0024] Figure 1 Strategies and maps of packaging constructs for single-cell AAV capsid and promoter library screening are described, according to some embodiments.

[0025] Figure 2Methods involving single-cell screening of AAV capsids and promoters according to some embodiments are described. The method is illustrated as an example in retinal tissue. Sub-figure A: A barcoded AAV library is injected into the tissue. Viral variants from the library infect different cells with different efficiencies. Sub-figure B: Efficient viruses enter cells, transfer to the nucleus, and lead to the expression of mRNA. Sub-figure C: Tissues are separated into single cells, and the mRNA of individual cells is labeled with cell-specific DNA barcodes. Sub-figure D: The transcriptome profile of individual cells is analyzed to determine the cell type, which AAV has infected the cell, and the specificity and efficiency of the AAV. Sub-figure E: For enhancer / promoter libraries, the library is packaged into a single AAV capsid with a wide range of tropisms. Sub-figure F: Different promoters drive different levels of gene expression in separate cell types. Sub-figure G: A single cell suspension is made, and the mRNA from individual cells is labeled with cell-specific DNA barcodes. Sub-figure H: The transcriptome profile of individual cells is analyzed to identify the cell type and determine the specificity and efficiency of the promoter.

[0026] Figure 3 An example of in vivo screening of AAV serotype constructed libraries in primate retina and brain is described. A library of 23 AAVs was individually packaged, and their genomes contained a ubiquitous promoter driving the expression of a green fluorescent peptide (GFP) fused to a unique DNA.

[0027] Figure 4 Described in Figure 3 Expression of GFP in the primate retina and brain after injection of the AAV library described in the description. These variants were packaged, combined and injected into the retina, prefrontal cortex (PFC) and striatum of rhesus monkeys. Injection of the library resulted in GFP expression in the retina and brain.

[0028] Figure 5 Data are shown for identification of intrinsically light-sensitive retinal ganglion cells (RGCs) in the rhesus macaque retina and recovery of barcodes from specific AAV serotypes. Each circle is an individual cell. Panel A shows OPN4 in the ICA space. + / - Cell clusters. Sub-figure B shows OPN4 + / - and POU4F2 + / - Identification of RGC cells. Larger circles indicate cells from which the AAV genomes were recovered (identified by their barcodes).

[0029] Figure 6is a heat map of AAV tropism for each cell type. Quantification of 23 existing serotypes showed that, as expected, AAV infectivity in the retina (K916, K94, 7m8, K912, NHP26) performed better than other variants. In contrast, AAV9-based vectors were the best performing variants in the putamen, validating the approach. AAV variants that did not infect the analyzed cell types are not shown in the heat map.

[0030] Fig. 7A is the AAV vector map of the library provided herein, which has no intervening sequence (IVS) between the minimal promoter and the small peptide sequence. Figure 7B is a map of AAV vectors of the library provided herein, which have an IVS between a minimal promoter and a small peptide sequence.

[0031] Figure 8 is an AAV vector map of the library, which contains an AAV promoter (P40 or P19+P40) to drive cap expression, and then a promoter (e.g., CAG promoter) driving the transgene within the ITR (e.g., CAG-GFP, CAG-GFP11, or CAG-split GFP with a membrane signal peptide).

[0032] Fig. 9 is the rep trans plasmid map, which contains the full-length rep sequence without the ITRs.

[0033] Fig.10 Contains cluster plots created from marmoset macula scATAC-seq data (using SnapATAC) and then integrated with scRNA-seq data from the same sample. Cell type predictions based on gene expression from the integrated scRNA-seq data (using Seurat). RHO = rod photoreceptor cell-specific gene; RGR = Muller glial cell-specific gene; and GRIK1 = Off bipolar cell-specific gene.

[0034] Fig.11A -C contains disease gene profiles identified for RS1 (Retinoschisin), USH2A (Usherin), and ABCA4 (ATP-binding cassette subfamily A member 4), respectively (upper left = rhesus monkey macula; lower left = marmoset macula; upper right = marmoset superior colliculus; lower right = marmoset inferior colliculus). DETAILED DESCRIPTION OF THE INVENTION

[0036] Provided herein are methods that include (a) creating a library of AAV mutants, wherein each AAV within the library contains a unique DNA sequence (barcode) that can be tracked to assess viral performance, (b) packaging the AAV variants or promoters into a packaging cell line using a dual (for capsid libraries) or triple (for some capsid and promoter libraries) transfection protocol, (c) injecting or otherwise introducing the AAV mutant library into a tissue in culture or one or more tissues of an animal host, (d) allowing the AAV mutant library sufficient time for the AAV vectors in the AAV mutant library to compete with each other in vivo (or in cultured tissues) and infect one or more tissues of the animal host into which the AAV mutants have been injected or otherwise introduced, and (e) using a single cell or single nucleus microfluidics approach to create a single cell or single nucleus cDNA library from cells within one or more tissues of the animal host into which the AAV mutant library has been injected.

[0037] In one embodiment, the library used in the methods provided herein is a (i.e., one or more) highly complex AAV mutant library. A map and cloning plan for making a highly complex library is shown in Figure 1 .

[0038] For example, where the library involves variant AAV capsids, the methods provided herein can provide a high-throughput method for creating AAV vectors that are highly efficient and / or specific for infecting targeted or multiple cell types. Similarly, where the library involves AAVs having variant upstream promoters operably linked to gene coding sequences, the methods provided herein can provide a high-throughput method for creating AAV vectors that are highly efficient and / or specific for achieving gene expression in targeted or multiple cell types.

[0039] AAV mutant libraries can be constructed so that each AAV within the AAV mutant library has a unique DNA barcode, e.g. Figure 1As shown in . In some cases, the Cap gene or promoter / enhancer, and the barcode are synthesized and cloned into the backbone, and then other sequences are cloned between the cap gene and the barcode to complete the packaging plasmid. The barcode is cloned into the same sample plasmid as the cap gene. The pairing between the AAV variant and the barcode, or the promoter and the barcode can be designed via computer simulation and then synthesized. Each AAV variant or promoter can be represented by one or more unique barcodes. These synthetic constructs are then cloned into the AAV packaging backbone. For the AAV capsid library, the backbone may include rep and cap sequences, (not included in the ITR sequence) so that the rep and cap genes are not packaged into the AAV capsid. On the same plasmid, the AAV genome includes a promoter that drives transgenic expression, such as a nucleic acid encoding GFP or other fluorescent polypeptides, fused with a unique DNA barcode, which can be located between the ITR packaging signals. Therefore, a single plasmid can include genes for producing AAV capsids, and a viral genome that includes a unique DNA sequence tag (barcode) that can be tracked to assess viral tropism and infectivity. The virus can then be packaged using, for example, a double transfection method, in which two plasmids: (1) rep / cap / transgene-barcode, and (2) a helper plasmid providing adenoviral helper functions, are transfected into packaging cells. In some cases, a triple transfection method is used, in which three plasmids ((1) rep plasmid, (2) promoter / cap / transgene-barcode plasmid, and (3) a helper plasmid providing adenoviral helper functions) are transfected into packaging cells. For capsid libraries that can be based on any AAV serotype, naturally occurring parental serotypes are also included in the library as a baseline against which the efficiency and specificity of AAV variants can be measured. For promoter libraries, the library construct can include a unique promoter that drives the expression of a transgene fused to a unique DNA sequence (barcode), by which the strength and specificity of the promoter can be assessed. In the case of a promoter library, the rep / cap gene can be provided in trans on another plasmid. In some cases, AAV can be packaged using a triple transfection method, in which three plasmids are transfected into the packaging cell line: (1) rep / cap plasmid, (2) promoter library-barcode construct, and (3) helper plasmid. For the promoter library, the ubiquitous CAG and CMV promoters can be synthesized and used as a baseline for measuring promoter efficiency and specificity.

[0040] Once constructed, the AAV mutant libraries provided herein can be packaged into packaging cell lines. For example, double (for capsid libraries) or triple (for some capsid libraries and promoter libraries) infection schemes can be used to package AAV variants or promoters. Any appropriate packaging cell line can be used, including but not limited to HEK-293 cells, HEK293T cells and AAV 293 cells.

[0041] Once constructed, the AAV mutant library can be injected or otherwise introduced into one or more tissues of a cultured tissue / organoid or animal host in vitro. The animal host can be any desired animal species that the AAV vector can infect, such as primates. Examples of primates used as animal hosts as described herein include, but are not limited to, New World Monkeys, Old World Monkeys (e.g., Rhesus Monkeys (Macaca mulatta)), Great Apes and Small Apes (e.g., Gibbons (gibbons)) Family or Hominidae (Bonobos, Chimpanzees, Gorilla, Orangutans)). In some cases, the host animal may not be of the genus Homo (late Homo sapiens (Homosapiens sapiens)) or of the genus Chimpanzee.

[0042] The tissue into which the AAV mutant library provided herein is injected or otherwise introduced can be any desired tissue, such as central nervous system (CNS) tissue (e.g., brain and spinal cord), peripheral nervous system tissue, retinal tissue, and any type of muscle tissue (e.g., striated muscle, cardiac muscle, smooth muscle tissue, etc.). Of course, other tissues / organs can be appropriately injected with the AAV mutant library provided herein, such as any internal or external tissue or organ (e.g., adrenal glands, bladder, colon, esophagus, external barrier tissue (skin, subcutaneous tissue, mucus-producing tissue, etc.), kidney, liver, lung, ovary, pancreas, rectum, small intestine, spleen, stomach, testis, thymus, ureter, etc.). In some cases, tissues grown in culture, such as retinal organoids, can be used as described herein.

[0043] In some cases, the methods provided herein can include allowing the AAV vectors in the AAV mutant library to have enough time to compete with each other and infect one or more tissues of the animal host (or cultured tissue) that has been injected or otherwise introduced into the AAV mutant library. This period of time will vary depending on the tissue and species of the host animal or the in vitro tissue source. In some cases, this period of time can be between about 1 week and about 12 weeks in a living organism and about 1-14 days in a cultured tissue. For example, after the AAV mutant library is introduced into a living animal host by injection or other means, the living animal host can be maintained for 1-12 weeks (e.g., 1-8 weeks, 1-5 weeks, 1-3 weeks, 3-10 weeks, 5-10 weeks, or 3-6 weeks) before being analyzed.

[0044] Subsequently, analysis can be performed to identify the best vector based on the presence and quantity of DNA bands in transcriptomes from many different cell types in parallel, based on, for example, specificity, expression level, and / or other desired properties (e.g., but not limited to, increased infectivity, increased specificity to one or more cell types, reduced immune response, etc.). In some cases, viral efficiency can be determined based on the number of GFP barcodes recovered from cells of a particular type. In some cases, vectors can then be ranked based on the level of transgene expression. Viral variants with the highest transgene expression levels compared to the most closely related parental serotype for a particular cell type can be considered the most efficient virus (best performer) for that cell type. Viral variants with the highest transgene expression levels compared to the most closely related parental serotype for a particular cell type, but with the lowest expression levels in other cell types, can be considered the most specific virus (best performer) for that cell type.

[0045] Selection can be performed at two levels: (1) highly diverse libraries of viral capsids can be screened for vectors with high efficiency and specific tropism, and (2) the ability of enhancer / promoter constructs to drive expression can be evaluated in specific cell populations. In this context, "high efficiency" refers to a virus that is more infectious for one or more cell types than a second virus. In addition, in this context, "specific" refers to a virus that is more infectious for one or more cell types than a second virus, while the infectivity or expression is reduced for all other cell types. In some cases, the performance of viral capsids can be evaluated based on mRNA transcript levels rather than DNA, which reflects the ability of the vector to drive the expression of the loaded protein, rather than just entering the cell. These steps can be performed in any species, including primate retina, brain or other tissues, to maximize the transformation potential of the resulting vector. In some cases, the high-throughput screening methods provided herein can allow the identification and characterization of viral variants and promoters with desired properties, including broad tropism and specificity.

[0046] The present invention is further illustrated by the following examples, which do not limit the scope of the present invention described in the claims. Example

[0047] Example 1 - Method for developing AAV vectors and promoters based on single-cell transcriptome

[0048] Materials and methods

[0049] Library synthesis

[0050] AAV libraries with (a) capsids or (b) promoters / enhancers were engineered to contain unique DNA barcodes. Multiple barcodes were available for each unique construct. Capsid libraries contained capsids with random mutations, semi-random peptide motifs, and random amino acid motifs spanning surface exposed positions and based on naturally occurring serotypes, naturally occurring serotype mixtures, or synthetic sequences. Enhancer / promoter libraries contained motifs and sequences taken from single cell ATAC-Seq experiments, synthetic sequences, and mutant versions of existing promoters.

[0051] To obtain direct correlation of AAV capsid variants and their barcodes, the cap sequence and the AAV genome were encoded on a single plasmid. Libraries were synthesized with the mutated cap gene directly upstream of the unique barcode, and the sequence between the mutated cap gene and the barcode was cloned between the two ( Figure 1 ). Variant-barcode pairings were reconfirmed by deep sequencing (high-throughput sequencing, such as ILLUMINA sequencing) before and after packaging.

[0052] For example, Figure 1 The strategy and map of packaging constructs for single-cell AAV capsid and promoter library screening are described. The cap gene or enhancer, and the barcode are synthesized and cloned into the backbone, and then other sequences are cloned between the cap gene and the barcode to complete the packaging plasmid.

[0053] For AAV capsid libraries, synthesizing libraries in this way allows the capsid and genome of the virus to be encoded in the same plasmid. Each HEK293 packaging cell transfected with the plasmid will package the virus, which contains a barcode specifying its unique capsid. Before each packaging, the optimal transfection MOI is determined by determining the minimum amount of library plasmid DNA required for sufficient packaging (the amount of DNA required to produce AAV titer> E+12vg / mL) to minimize or prevent cross-packaging. Multiple barcodes for each serotype are included to ensure that the background noise of any potential cross-packaging is reduced. Enhancers are cloned into a skeleton containing a known minimal promoter that drives different levels of expression (for example, but not limited to minimal cytomegalovirus (DMV), heat shock protein 68 (HSP68), GATA binding factor 2 (GATA2) and sterol transporter protein (SCP2)) or a promoter sequence associated with the identified enhancer determined by calculation. Different minimal promoters are identified by other 3 base pair tags present in the skeleton.

[0054] Single-cell screening of AAV capsids and promoters

[0055] To assess the performance of each member of the capsid and promoter library, scRNA-Seq was used to identify cell types and quantify vector and promoter efficiency and specificity, as Figure 2as shown in .

[0056] A capsid library containing each capsid has a unique barcode injected, and the vectors then infect cells with different tropisms, efficiencies, and specificities. Single-cell suspensions are created from injected tissues, and cells are identified by transcriptome profiling. In parallel, capsid performance is quantitatively assessed by the number of GFP-barcoded transcripts recovered from the variants. Promoter libraries are packaged into separate variants with broad tropisms, and each promoter is paired with a unique barcode. Viruses packaged with enhancer library members infect cells, and specificity and efficiency are quantified by counting GFP-barcoded transcripts in each cell type after scRNA-Seq.

[0057] Figure 2 Summarize the exemplary method of AAV capsid and promoter related to single cell screening provided herein, use retinal tissue as example.For sub-figure A, barcoded AAV library is injected into tissue.Viral variants from the library infect different cells with different efficiencies.For sub-figure B, efficient virus enters cells, transfers to the nucleus, and causes the expression of mRNA.For sub-figure C, tissue is separated into single cells, and the mRNA of single cells is labeled with cell-specific DNA barcodes.For sub-figure D, the transcriptome profile of single cells is analyzed to determine the cell type, and which AAV has been infected with the cell, and the specificity and efficiency of AAV.For sub-figure E, for enhancer / promoter library, the library is packaged into a single AAV capsid with a wide range of tropism.For sub-figure F, different promoters drive different levels of gene expression in separate cell types.For sub-figure G, single cell suspension is made, and the mRNA from single cells is labeled with cell-specific DNA barcodes.For sub-figure H, the transcriptome profile of single cells is analyzed to identify cell types and determine the specificity and efficiency of promoters.

[0058] Quantitative comparison of the best performing capsid and promoter subsets

[0059] To verify the performance of the best candidates for capsid and enhancer, a second round of evaluation was performed. The best vectors targeting specific cell types were selected (viruses with the highest level of transgene expression compared to the parental serotype, or viruses with the highest level of transgene expression and the lowest level of transgene expression in all other cell types compared to the parental serotype). For each cell type or gene, the most efficient virus (the variant that drives the most copies of the barcode transcript, normalized by the total number of transcripts per cell) and the variant that drives the most specific and efficient expression (the variant that drives the most copies of the on-target barcode transcript and the lowest copies of the off-target transcript) and the variant that drives the gene expression pattern that best matches the wild-type expression pattern of the pathogenic gene were selected. Variants with different levels of off-target expression were selected for secondary screening to determine the optimal threshold for off-target readings. These variants will be packaged again with unique barcodes (for example, but not limited to GFP-fusion barcodes), pooled, injected into tissues, and screened again by scRNA-Seq to determine the overall best performing vector.

[0060] The best performing capsids and promoters were then paired and validated separately. The performance of the best candidate vectors and promoters was quantified and ranked, and the overall best performers were identified for each cell type. Expression profiles were further assessed in the retina and brain by in vivo imaging, histology, qRTPCR, and scRNA-Seq.

[0061] Therefore, based on the experiments presented here, the single-cell approach to AAV screening described here can be used to evaluate the performance of AAV vectors in different cell types. In the retina, intrinsically photosensitive retinal ganglion cells (ipRGCs) were identified as a subpopulation of RGCs, and barcodes were recovered from these cells ( Figure 5 ). Single-cell RNA-seq analysis of AAV serotypes in retinal and brain cells showed that, as expected, the retinal-evolved serotypes performed best in the retina, while AAV9 and AAV92YF performed best in putamen neurons ( Figure 6 ).

[0062] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference in its entirety.

[0063] The terms "one" and "an" and "the" and "at least one" and similar expressions used in describing the present invention (especially in the context of the appended claims) should be interpreted as covering the singular and the plural, unless otherwise stated or the context clearly indicates otherwise. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be interpreted as indicating one item selected from the listed items (A or B) or any combination of two or more listed items (A and B), unless otherwise stated herein or clearly contradicted by the context. The terms "comprising", "having", "including" and "comprising" should be interpreted as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated herein. Unless otherwise stated herein, references to numerical ranges herein are merely a shorthand method of indicating that each independent value falling within the range is individually indicated, and each independent value is included in the specification as if stated separately herein. All methods described herein can be performed in any suitable order, unless otherwise stated or clearly indicated to the contrary. Any and all embodiments / examples, or the use of exemplary language (e.g., "for example / such as / such as") referred to herein are merely for better illustrating the present invention, and are not intended to limit the scope of the present invention, unless otherwise specified. All language in the specification should not be construed as indicating non-claimed elements that are essential to the practice of the present invention.

[0064] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. By reading the foregoing description, variations of those preferred embodiments will become apparent to those of ordinary skill in the art. The inventor expects that skilled technicians will appropriately adopt such variations, and the inventor wishes to practice the present invention in a manner different from that specifically described herein. Therefore, many embodiments included in the present invention include all modifications and equivalents of the subject matter described in the appended claims as allowed by applicable law. Moreover, unless otherwise noted herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof.

[0065] Example 2 - AAV library

[0066] Other versions of the AAV library were constructed so that the AAV uses the native conformation of the AAV genome and places an additional small peptide within the genome ( Fig. 7A and 7B Briefly, the wild-type AAV genome is retained within the ITR packaging signal. A small peptide tag and a small minimal pA signal (e.g., a 48 bp poly A signal) driven by a small ubiquitous promoter (e.g., a mini-CMV promoter) are added after the cap open reading frame and between the ITRs ( Fig. 7AThe library may contain an intervening sequence (IVS) between a minimal promoter and a small peptide sequence ( Figure 7B ).

[0067] Example 3 - AAV library

[0068] Other versions of the AAV library were constructed so that the strong ubiquitous promoter drives the expression of GFP or split GFP (split GFP can be displayed on the cell surface or retained in the cell cytoplasm) and so that the cap gene is packaged inside the ITR ( Figure 8 In these cases, libraries with rep are created in trans ( Fig. 9 ), which has the added benefit of being able to generate a replication-incompetent library to increase safety in animals that may have helper virus infection, but allows the cap to be driven by the endogenous AAV promoter and the cap to be packaged inside the virus along with a barcode representing a series of amino acid insertions in the cap gene. Briefly, these libraries can contain the AAV P40 promoter, the AAV P19+P40 promoter, or a longer version of the AAV P19+P40 promoter. The construct can also contain a promoter, such as the ubiquitous CAG promoter, that drives expression of a fluorophore, such as GFP, GFP11 (e.g., split GFP), or GFP11 expressed on the cell surface.

[0069] Example 4 - Designing libraries for cell type specific promoters using scATAC-Seq data

[0070] Construction of promoter libraries from scATAC datasets, e.g. Fig.10 Datasets used to cluster single cells as shown in . Single-cell ATACseq was used to identify open chromatin regions in isolated retinal cells. Cluster plots were created from marmoset macula scATAC-seq data (using SnapATAC) and then integrated with scRNA-seq data from the same sample. Cell types were predicted based on gene expression from the integrated scRNA-seq data (using Seurat). Promoter libraries were constructed by co-pairing multiple DNA sequences from specific cell types.

[0071] Example 5 - AAV selection for disease-specific AAV

[0072] The profile of the disease gene can be determined by single-cell RNA-Seq, and then the desired AAV (providing the natural expression pattern and expression level of the wild-type copy of the gene) can be determined by matching the AAV profile to the disease gene profile. The disease gene profile is for RS1, USH2A, and ABCA4 (respectively Fig.11A , 11B and 11C) determined.

[0073] Other Implementations

[0074] It should be understood that although the invention has been described in conjunction with the specific description, the above description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages and improvements are also within the scope of the following claims.

Claims

1. A method comprising: (a) creating a library of AAV mutants, wherein each AAV in the library contains a unique DNA barcode, (b) packaging of AAV mutants into packaging cell lines using a double transfection protocol, (c) delivering the AAV mutant library into one or more tissues of an animal host, wherein the animal host is a primate that is not a human, (d) maintaining the AAV mutant library in vivo for a period of time suitable for the AAV vectors in the AAV mutant library to compete with each other in one or more tissues of an animal host to which the AAV mutant library has been delivered, wherein the period of time is at least one week, and (e) obtaining tissue from the animal host into which the AAV mutant library has been delivered, (f) isolating tissue obtained from the animal host to obtain single cells, and (g) analyzing the transcriptome profile of the single cell to identify (i) the cell type of the single cell and (ii) the AAV mutant that infected the single cell based on the unique DNA barcode of the AAV mutant.

2. The method of claim 1, wherein the one or more tissues of the animal host include neural tissue.

3. The method of claim 2, wherein the neural tissue comprises central nervous system tissue.

4. The method of claim 3, wherein the central nervous system tissue is brain tissue.

5. The method of claim 2, wherein the neural tissue comprises peripheral nervous system tissue.

6. The method of claim 1, wherein the one or more tissues of the animal host include retinal tissue.

7. The method of claim 1, wherein the one or more tissues of the animal host include muscle tissue.

8. The method of claim 7, wherein the muscle tissue comprises striated muscle.

9. The method of claim 7, wherein the muscle tissue comprises cardiac muscle.

10. The method of claim 7, wherein the muscle tissue comprises smooth muscle.

11. The method of claim 1, wherein the primate is an Old World monkey.

12. The method of claim 11, wherein the Old World monkey is a rhesus monkey (Macaca mulatta).

13. The method of claim 1, wherein the primate is an ape of the family Gibbon or Hominidae.

14. The method of claim 1, wherein the primate is not a chimpanzee.

15. The method of any one of claims 1-14, wherein delivery of the AAV mutant library is by injection into the tissue.

16. A method for obtaining an AAV mutant having the ability to infect a desired cell type in vivo and to persist in vivo in said cell type for at least one week, wherein said method comprises: (a) introducing a library of AAV mutants into an animal host comprising said cell type, wherein each AAV in said library comprises a unique DNA barcode, wherein said animal host is a primate, and said primate is not a human, and (b) maintaining the AAV mutant library in vivo for a period of time suitable for AAV vectors in the AAV mutant library to compete with each other, wherein the period of time is at least one week, (c) obtaining a tissue comprising said cell type from said animal host, (d) isolating tissue obtained from the animal host to obtain single cells, and (e) analyzing the transcriptome profile of the single cell to identify (i) the single cell and (ii) the AAV mutant that infects the single cell based on the unique DNA barcode of the AAV mutant.

17. The method of claim 16, wherein the cell type is a central nervous system cell type or a peripheral nervous system cell type.

18. The method of claim 16, wherein the cell type is a retinal cell type, a striated muscle cell type, a cardiac muscle cell type, or a smooth muscle cell type.

19. The method of claim 16, wherein the primate is an Old World monkey.

20. The method of claim 16, wherein the primate is a rhesus monkey (Macaca mulatta).

21. The method of claim 16, wherein the primate is an ape of the family Gibbon or Hominidae.

22. The method of claim 16, wherein the primate is not a chimpanzee.

23. The method of any one of claims 16-22, wherein the library is introduced into the animal host by injection into a tissue containing the cell type.

24. The method of any one of claims 16-22, wherein the at least one week is from 1 week to 12 weeks.

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