Methods for directed evolution of AAV capsid using transcriptional dependency

Abstract

The present invention discloses methods of performing transcriptional dependent directed evolution (TRADE) and novel AAV capsids selected using such methods. The invention also provides a novel AAV capsid mutant. The TRADE technology is used to identify novel AAV vectors that mediate neuronal transduction in the brain following intravenous administration. The use of TRADE in vivo results in the identification of novel AAV capsids that, after administration of the novel AAV capsids, can transduce neurons more efficiently, more specifically in the brain than AAV9. The TRADE technique can be used to identify AAV capsids that target various cell populations.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of January 31, 2020, application number 202080026983.X, and invention name “Method for using transcription-dependent directed evolution of AAV capsids” (the application date of the corresponding PCT application is January 31, 2020, and the application number is PCT / US2020 / 016273). Technical Field

[0002] The present disclosure relates to viral vectors for gene delivery. More specifically, the present disclosure relates to methods for transcription-dependent directed evolution and adeno-associated virus ("AAV") vectors selected by using the method. Background Art

[0003] Recombinant adeno-associated virus ("AAV") vectors are one of the most promising vectors for in vivo gene delivery. Since many naturally occurring new serotypes and subtypes have been isolated from human and non-human primate tissues, the use of AAV vectors has been expanded. Gao et al., J Virol 78, 6381-6388 (2004) and Gao et al., Proc Natl Acad Sci USA 99, 11854-11859 (2002). Among the newly identified AAV isolates, AAV serotype 8 (AAV8) and AAV serotype 9 (AAV9) are of interest because AAV vectors derived from these two serotypes can transduce multiple organs with high efficiency after systemic administration, including liver, heart, skeletal muscle and central nervous system. Ghosh et al., Mol Ther 15, 750-755 (2007); Pacak et al., Circ Res 99, 3-9 (2006); Inagaki et al., Mol Ther 14, 45-53 (2006); Zhu et al., Circulation 112, 2650-2659 (2005); Wang et al., Nat Biotechnol 23, 321-328 (2005); Nakai et al., J Virol 79, 214-224 (2005); and Foust et al., Nature Biotechnol 23, 321-328 (2009). This potent transduction of AAV8 and AAV9 vectors is attributed to the strong tropism for these cell types, efficient cellular uptake of the vectors, and / or rapid uncoating of the virion capsid in the cells. Thomas et al., J Virol 78, 3110-3122 (2004). In addition, the emergence of AAV vectors with better capsid engineering has significantly expanded the utility of AAV as a vector toolkit. Asokan et al., Mol Ther 20, 699-708 (2012).

[0004] Proof of concept using AAV-mediated gene therapy has been demonstrated in many preclinical animal models of human disease. Phase I / II clinical studies have shown promising results for the treatment of hemophilia B (Nathwani et al., N Engl J Med 71, 1994-2004 (2014)), lipoprotein lipase deficiency (Carpentier et al., J Clin Endocrinol Metab 97, 1635-1644 (2012)), Leber congenital amaurosis (Jacobson et al., Arch Ophthalmol 130, 9-24 (2012) and Pierce and Bennett, Cold Spring Harb Perspect Med 5, a017285 (2015)) and others (reviewed in Mingozzi and High, Nat Rev Genet 12, 341-355 (2011) and Wang et al., Nat Rev Drug Discov 18, 358-378 (2019)).

[0005] Despite this hope, human studies have also revealed unexpected problems and potential challenges in AAV-mediated gene therapy. Manno et al., Nat Med 12, 342-347 (2006). In addition, despite our rapid progress in understanding the relationship between AAV biology and capsid phenotypes (Adachi et al., Nat Commun 5, 3075, (2014); Grimm et al., Hum Gene Ther 28, 1075-1086, (2017); and Ogden et al., Science 366, 1139-1143, (2019)), for clinical AAV vectors, there are still many desirable properties that we cannot rationally design.

[0006] To this end, high-throughput screening methods have been used to identify novel AAV capsids with such desired phenotypes. In particular, the development of in vivo AAV library selection strategies has produced a variety of designed AAV variants that can efficiently transduce previously intractable cell types (Kotterman and Schaffer, Nat Rev Genet 15, 445-451 (2014) and Grimm et al., Mol Ther 23, 1819-1831 (2015) reviewed).

[0007] The earliest attempts at in vivo library selection (first generation) relied on the recovery of vector genomic DNA from dissected tissue. In theory, this strategy would result in the recovery of potent AAV variants as well as AAV variants that mediate some but not all steps required for vector-mediated transgene expression ( Figure 1). Therefore, screening a diverse library of synthetic AAV variants may result in high background recovery of AAV variants that are completely ineffective gene therapy vectors. In addition, further processing is required for specific cell types, such as fluorescence-activated cell sorting or laser capture microdissection. Nevertheless, there have been some reports of successful use of this technology. Excoffon et al., Proc Natl Acad Sci USA 106, 3865-3870 (2009); Grimm et al., J Virol 82, 5887-5911 (2008); Lisowski et al., Nature 506, 382-386 (2014); and Dalkara et al., Sci Transl Med 5, 189ra176 (2013). However, a landmark study by Deverman et al. in 2016 showed that this process can be greatly improved by using a Cre-dependent selection strategy (second generation). Deverman et al., Nat Biotechnol 34, 204-209 (2016). Cre-dependent library selection utilizes the selective ability of Cre recombinase to act on double-stranded DNA instead of single-stranded DNA to invert the vector genomic DNA containing primer binding sequences. The inversion of this sequence allows direction-selective PCR to specifically amplify the viral DNA delivered to cells by AAV variants, which can undergo the late transduction period, at which stage double-stranded DNA is formed from the single-stranded AAV genome. In addition, the use of Cre driver lines helps to selectively express Cre recombinase in a cell type-specific manner, thereby allowing the selection of novel AAV variants for effective transduction. In fact, the use of Cre-dependent selection enables the authors to develop an AAV9 variant AAV-PHP.B, which, after systemic administration to C57BL / 6J mice, has a transduction capacity 40 times that of the parental AAV9. Deverman et al., Nat Biotechnol 34, 204-209 (2016). Unfortunately, it has recently become clear that the enhancement shown by AAV-PHP.B in mice has not been transformed into the environment of non-human primates (Matsuzaki et al. 2018 and Hordeaux et al. 2019). Surprisingly, this enhancement does not even extend to all commonly used mouse strains (Matsuzaki et al. 2018 and Hordeaux et al. 2019). Therefore, there is a strong impetus to accelerate the development of clinically relevant AAV vectors by conducting AAV library selection experiments in primate models. However, unlike AAV variant selection in mice, for which a plethora of cell-type-specific transgenic Cre-driven lines have been established, Cre-dependent selection is difficult to harness in clinically relevant large animals, including nonhuman primates, because Cre transgenic animals are not readily available.

[0008] Therefore, we seek to develop a next-generation selection strategy (generation 3) that has similar or better selection stringency than that provided by Cre-dependent selection and does not require Cre recombinase. To achieve this goal, we developed a transcription-dependent directed evolution (TRAnscription-dependent Directed Evolution) system, i.e., TRADE. In transcription-dependent selection, we express the AAV cap gene as a non-coding antisense mRNA driven by a cell type-specific enhancer-promoter. Recovering this antisense transcript by RT-PCR allows strict recovery of the AAV cap gene at a vector-mediated mRNA expression level in a specific cell type without the need to use Cre recombinase. Targeting different cell types only requires cloning different cell type-specific enhancers-promoters into plasmid constructs. Therefore, TRADE is a highly flexible system that can be applied to a variety of environments, including non-human primate environments for the development of enhanced AAV vectors for clinical gene therapy. Please note that the same principle can be used to express the AAV cap gene in the sense direction. However, the sense strand method results in the expression of immunogenic capsid proteins in target cells, and is therefore not as ideal as the antisense strand method used by the TRADE system. Summary of the invention

[0009] The present disclosure provides a next generation directed evolution strategy called transcription-dependent directed evolution ("TRADE"), which selects AAV capsid transduction at cell type specificity or ubiquitous mRNA expression levels. The method described herein provides the following advantages compared with AAV targeted evolution ("CREATE") based on Cre recombination, which is the most modern method of AAV capsid directed evolution reported in the literature. Deverman et al., Nat Biotech 34,204-209 (2016). First, the CREATE system requires Cre expression, which can be achieved by exogenously delivered Cre expression or using Cre transgenic animals. In contrast, the TRADE system does not require Cre transgenic animals; therefore, it can be applied to animals and cultured cells derived from any animal species, and can be easily adapted to large animals, including non-human primates. Secondly, in the CREATE system, cell type specific selection is applied at the conversion level of the AAV viral genome from single-stranded DNA to double-stranded DNA, and unlike the CREATE system, TRADE allows cell type specific selection at the AAV genome transcription level. Therefore, the TRADE system can provide greater selection pressure than the CREATE system. Third, multiple directed evolution schemes (e.g., neuron-specific, astrocyte-specific, oligodendrocyte-specific, and microglia-specific) can be integrated into one AAV capsid library, and AAV vectors targeting each cell type can be selected in a single animal. Fourth, any cell type-specific or tissue / organ-specific enhancer / promoter or ubiquitous enhancer / promoter can be easily used for AAV capsid directed evolution, aiming to identify cell type-specific or ubiquitous novel AAV capsids with enhanced efficacy. Fifth, the TRADE method is not limited to the Dependoparvovirus genus, including common AAVs that have been used for gene delivery, but can also be more broadly applied to the Parvoviridae family, including the genera Bocaparvoviruses and Erythroparvoviruses (e.g., Bocavirus) in addition to AAV, and more broadly to DNA viruses.

[0010] The present disclosure also provides novel AAV capsid mutants. TRADE technology is used to identify novel AAV vectors that mediate neuronal transduction in the brain after intravenous administration. The application of TRADE in C57BL / 6J mice and rhesus monkeys has led to the identification of new AAV capsids that can transduce neurons in the brain of mice and non-human primates more efficiently and specifically than AAV9 after intravenous administration. In addition, we have identified novel AAV capsids that can transduce one or more undefined cell populations present in the lungs and that may be of neuronal origin 5 to 18 times better than AAV9.

[0011] The present disclosure also provides methods for preventing splicing of antisense mRNA of AAV capsid genes. Antisense precursor mRNA transcribed from the AAV cap gene open reading frame ("ORF") can be spliced ​​to produce truncated mRNA species. To our knowledge, this is a new discovery that has never been reported before. This splicing has the potential to hinder the efficient recovery of full-length antisense mRNA of the AAV cap ORF, which is essential for TRADE when wide regions of the cap ORF are mutagenized. The present disclosure provides a new strategy to prevent splicing of antisense mRNA of the cap gene.

[0012] The TRADE system described herein uses antisense mRNA to recover capsid sequence information, and TRADE using sense strand mRNA (i.e., sense strand TRADE) is also feasible using the same principle. However, it should be noted that the sense strand TRADE method results in the expression of immunogenic capsid proteins in target cells and may therefore be less ideal than the antisense strand method. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Overview of in vivo library selection strategies for directed evolution of AAV capsids. AAV vector-mediated transduction is a multistep process that requires the virion to overcome the extracellular barrier, bind to receptors on target cells, enter cells by endocytosis, escape endosomes, transport to the nucleus, uncoat, achieve double-stranded DNA configuration, and finally undergo transcription / translation. The earliest in vivo library selection strategies (first generation) recovered all vector genomic DNA from tissue samples. In theory, this strategy will recover effective AAV variants, as well as AAV variants that mediate some but not all steps required for vector-mediated transgene expression. In addition, this strategy can also recover AAV vector genomic DNA from AAV vector particles that do not enter cells and remain in the extracellular matrix. Therefore, screening a diverse library of synthetic AAV variants will result in relatively high background recovery of AAV variants that are completely ineffective gene therapy vectors. In addition, focusing on specific cell types requires further processing, such as fluorescence-activated cell sorting (FACS) or laser capture microdissection (LCM). Second generation library selection (Generation 2) significantly improves selection stringency by utilizing Cre-dependent recovery of only those AAV variants that can achieve the double-stranded DNA stage of transduction. In addition, driving Cre expression with cell type-specific enhancers-promoters allows targeting of specific cell types while retaining the benefits of processing a large number of tissue samples. Third generation library selection (Generation 3) is further based on the AAV directed evolution technology of transcription-dependent recovery using AAV variants, which can mediate transgenic mRNA expression from cell type-specific enhancers-promoters without Cre expression.

[0014] Figure 2TRADE principles. Figure 2 Figure A is a diagram of the AAV vector genome in the TRADE configuration (AAV-TRADE). The cell type specific enhancer-promoter is placed in the antisense direction to drive the AAV cap gene transcriptional expression as antisense mRNA. The polyadenylation signal (pA) derived from the simian virus 40 (SV40) genome is placed in the AAV genome intron in the antisense direction to terminate the antisense AAV cap gene mRNA transcription. The eGFP open reading frame (ORF) can be placed as shown in the figure to serve as a reporter or to promote the enrichment of transduced cells by FACS; however, such a marker gene is not absolutely necessary for TRADE. Ubiquitous promoters (such as CAG promoters) can also be used in TRADE to replace cell type specific enhancers-promoters to identify AAV capsids that can transduce multiple cell types. The cell type specific enhancer-promoter can be placed upstream of the AAV cap gene ORF to drive the expression of AAV cap gene mRNA transcripts in the sense direction (i.e., sense strand TRADE). However, this approach may not be ideal for TRADE because AAV capsid proteins will be expressed in target cells, which may lead to undesirable biological outcomes during directed evolution. Figure 2 As shown in Figure B, during the preparation of AAV vectors in HEK293 cells, in the presence of adenoviral helper functions, the AAV2 viral p40 promoter drives cap gene expression (forward transcription) and cell type-specific transcripts are repressed, resulting in the successful preparation of recombinant AAV vectors containing the AAV-TRADE vector genome. Upon transduction of a specific cell type, the cell type-specific enhancer-promoter is activated to drive the expression of eGFP and antisense cap mRNA sequences, while the transcriptional activity of the p40 promoter remains inactive in the transduced cells due to the lack of adenoviral helper functions. The entire cap gene ORF can be recovered by reverse transcription (RT)-PCR using antisense cap gene mRNA expressed in a cell type-specific manner as a template. We have observed that recombinant AAV vectors can be successfully prepared at high levels even in the presence of antisense mRNA transcripts expressed due to leaky expression of the human synapsin I gene (hSynI) enhancer-promoter in HEK293 cells. We also observed that recombinant AAV vectors could be successfully prepared at high titers even when we used the CAG promoter that drives high-level expression of antisense AAV cap mRNA transcripts.

[0015] Figure 3 Validation of the TRADE system targeting brain neurons. Figure 3 Figure A is a diagram of the AAV-PHP.B-hSynI-GFP-TRADE vector genome. Figure 3As shown in Figure B, to validate the TRADE system, the AAV vector genome was packaged into AAV-PHP.B capsid as a single-stranded DNA genome, and the resulting AAV vector was transfected at 3 × 10 cells per mouse. 11 A dose of 1 vector genome (vg) was intravenously injected into two 8-week-old C57BL / 6J mice. Brain tissue was harvested 12 days after injection. Brain tissue from one animal was fixed with 4% paraformaldehyde for immunofluorescence microscopy, and brain tissue from another animal was not fixed for molecular analysis of AAV vector genomic DNA and RNA. Figure 3 C shows an immunofluorescence microscopy image of a brain section stained with an anti-GFP antibody confirming the expression of cell type-specific enhancer-promoter driven transcripts. Figure 3 Middle D shows that hSynI enhancer-promoter driven GFP expression was specifically observed in neurons (anti-HuC / D+). Figure 3 Figure E shows RT-PCR for recovering the full-length cap ORF sequence (RT+). RT-, no reverse transcriptase control; Plas, positive control obtained by DNA-PCR using a plasmid template containing the AAV-PHP.B-hSynI-GFP-TRADE vector genome sequence; NT, no template PCR. Figure 3 As shown in Figure F, Sanger sequencing of the RT-PCR product shows the expected splicing of the MVM intron in the antisense transcript expressed by the hSynI enhancer-promoter (SEQ ID NO: 190). Exon-exon junctions are highlighted in gray. Figure 3 As shown in G, Sanger sequencing confirmed the insertion of the PHP.B peptide (highlighted in grey) (SEQ ID NO: 191).

[0016] Figure 4 Splicing of AAV9 cap ORF antisense mRNA. Two cell lines, HEK293 and Neuro2a, were transfected with plasmids containing the AAV9 cap ORF in the TRADE configuration with or without the GFP reporter gene. They are represented in the figure as "GFPTRADE" and "TRADE", respectively. Cells were harvested 3 days after transfection, RNA was extracted, and RT-PCR was performed using a set of PCR primers that amplify the full-length cap ORF sequence. We did not recover the expected amplicon size of 2.4 kb, as shown in the positive control (PC) lane, we consistently recovered an amplicon of approximately 0.7 kb. Sanger sequencing of these RT-PCR products identified the Figure 5Splice consensus truncations of the 1.7 kb region of the AAV9 cap ORF are shown. PC, positive control using a plasmid template containing the AAV-PHP.B-hSynI-GFP-TRADE vector genome sequence; NC, no-template PCR control.

[0017] Figure 5 Intron identified in antisense mRNA derived from AAV9cap gene (SEQ ID NO: 192). When AAV-PHP.Bcap gene sequence was transcribed in antisense orientation in HEK293 cells or Neuro2a cells under the control of neuron-specific human synapsin I (hSynI) enhancer-promoter, splicing events and cryptic splice donor and splice acceptor sites were identified (see also Figure 6 ). The underlined sequence represents an intron found within the AAV9 cap ORF. This splicing event has not been observed in mouse brain neurons. It should be noted that (1) although the hSynI enhancer-promoter has been used as a neuron-specific element, it has been shown to drive leaky expression in HEK293 cells; (2) the AAV9 cap ORF sequence used for intron splicing experiments has the following silent mutation near the C-terminus: gaaccccgccccattggcacGCgTtacCTGACTCGTAATCTGTAA (SEQ ID NO: 1). Intron sequences are underlined, and silent mutations that have been introduced into the intron to create an MluI (ACGCGT) recognition site are indicated in uppercase letters.

[0018] Figure 6 Cryptic splice donor (SD) and splice acceptor (SA) sites with common features of exon-intron junctions present in the AAV cap ORF in the antisense orientation. Nucleotide sequences of cap genes derived from 122 naturally occurring AAV strains (serotypes and variants) were aligned using a multiple sequence alignment program (SEQ ID NOs 223-316). Exon-intron junctions identified in the AAV9 cap ORF-derived antisense mRNA are represented by solid lines. Dashed lines in the splice acceptor region represent putative splice acceptor sites in the AAV cap ORF, which do not contain the splice acceptor AG / TC sequence at the position expected from sequence conservation. Dashed lines in the splice donor region represent splice donor sites identified in the AAV3 cap ORF-derived antisense mRNA (see Figure 7). The GT / CA splice donor site and the AG / TC splice acceptor motif, followed by a run of Ts, are common features of exon-intron junctions and are well conserved in many AAV strains. The splice donor and splice acceptor sites identified in the AAV9 cap ORF shown in this figure have also been identified in the AAV1 cap ORF. For serotypes other than AAV1, AAV3, AAV5, and AAV9, splicing events in the antisense mRNA of the AAV cap ORF are currently being investigated. The highlighted variants are common AAV serotypes.

[0019] Figure 7 Introns identified in antisense mRNA derived from the AAV3 cap gene. pAAV3-hnLSP-MCS-TRADE2 is a plasmid carrying the wild-type AAV3 cap ORF with the MVM intron (hnLSP) placed under a liver-specific enhancer-promoter. The nucleotide sequence of the AAV3 cap ORF is identical to that of the naturally identified AAV3. HepG2 cells, a human hepatoma cell line, were transfected with the plasmid pAAV3-hnLSP-MCS-TRADE2. Antisense mRNA derived from the AAV3 cap ORF was then analyzed by RT-PCR. The sequences of the two truncated RT-PCR products were determined by Sanger sequencing after blunt-end TOPO cloning of the PCR products, which showed the introns found within the antisense AAV3 cap ORF ( Figure 7 (A) and (B) in SEQ ID NO: 193). Intron sequences are underlined lowercase letters. The upstreammost splice donor site was found to be only 3 bp away from the splice donor site identified in the AAV9 cap ORF, as shown in Figure 6 The most downstream splice acceptor site was found to be approximately 80 bp upstream of the splice acceptor site of the AAV9 cap ORF. Note that all splice donor and splice acceptor sites identified in the AAV3 cap ORF have also been identified in the AAV1 cap ORF.

[0020] Figure 8A-B . Other cryptic splice acceptor sites present in the AAV cap ORF. ( Fig. 8A and Figure 8B ) The nucleotide sequences of cap genes derived from 122 naturally occurring AAV strains (serotypes and variants) (SEQ ID NOs: 317-420) were aligned using a multiple sequence alignment program. Exon-intron junctions at splice acceptor sites identified in the AAV3 cap ORF-derived antisense mRNA are indicated by solid lines. Fig. 8AThe dashed line in the figure indicates an alternative putative splice acceptor site near the experimentally determined splice acceptor site. The AG / TC splice acceptor site, followed by a run of Ts, is a common feature of exon-intron junctions at splice acceptor sites and is well conserved in many AAV strains. The AAV3 cap ORF is highlighted. It has also been identified in the AAV1 cap ORF. Fig. 8A and Figure 8B The splice acceptor sites identified in the AAV3 cap ORF are shown in Figure 2. As for the AAV5 cap ORF, no splicing events were observed at any site in the antisense mRNA transcript. For serotypes other than AAV1, AAV3, AAV5, and AAV9, splicing events in the antisense mRNA of the AAV cap ORF are currently being investigated.

[0021] Fig. 9 Other potential splice donor sites present in the AAV cap ORF. The nucleotide sequences of the cap genes derived from 122 naturally occurring AAV strains (serotypes and variants) were aligned using a multiple sequence alignment program (SEQ ID NOs: 421-461). The exon-intron junction at the splice donor site identified in the antisense mRNA derived from the AAV3 cap ORF is indicated by a solid line. The GT / CA splice donor consensus sequence at this position is retained by only half of the AAV strains. This splice donor site has been identified in the AAV1 cap ORF.

[0022] Fig.10Splice donor and splice acceptor sites identified in AAV1 cap ORF. The nucleotide sequence of AAV1 cap ORF is shown (SEQ ID NO: 194). AAV1 cap ORF was expressed in antisense orientation in human embryonic kidney (HEK) 293 cells or Neuro2a cells by hSynI enhancer-promoter. Antisense mRNA derived from AAV1 cap ORF was then analyzed by RT-PCR. The sequence of the RT-PCR product was determined by Sanger sequencing after blunt-end TOPO cloning of the PCR product, which shows the introns found in AAV1 cap ORF. The exon-intron junction identified in the antisense AAV1 cap mRNA represents the splice donor site with AG / TC and the splice acceptor site with GT / CA. Capitalized AG / TC and GT / CA are two common nucleotides at the 5' and 3' ends of the intron, respectively. Since splicing occurs in the antisense mRNA of the ORF, the intron sequence is located between CT (splice acceptor) and AC (splice donor) in various combinations in the above sequence. Detailed information about the observed splice donor and acceptor combinations is not shown. The two conserved nucleotides at the exon-intron junction (CT or AC) indicated in bold are those that are highly conserved in different AAV serotypes. The two conserved nucleotides at the exon-intron junction (CT or AC) underlined are those that have also been identified in antisense AAV3 or AAV9 cap mRNA transcripts.

[0023] Fig.11 Splicing inhibition mutagenesis of AAV9 cap ORF. Silent mutations were introduced around the splice acceptor (SA) site and / or splice donor (SD) site in the AAV9 cap ORF to inhibit splicing observed on antisense mRNA transcripts. Introns spliced ​​out from the native sequence (SEQ ID NO: 195, SEQ ID NO: 196) are underlined. The AAV9NS1 genome (SEQ ID NO: 197) has one set of mutations around the SA site, while the AAV9NS2 genome (SEQ ID NO: 198) has one set of mutations around the SD site. The AAV9NS3 genome has two sets of mutations. The numbers on the right indicate the nucleotide position relative to the first nucleotide of the AAV9 cap ORF.

[0024] Fig.12Mutations introduced around the splice donor and / or acceptor sites effectively inhibited the splicing of antisense mRNA derived from the AAV9 cap ORF. Neuro2a cells were transfected with plasmids containing the AAV9 cap ORF and various potential splice inhibitory mutations (NS1-3) in TRADE configurations. RNA was harvested 3 days after transfection and RT-PCR was performed using a set of PCR primers that can recover the full-length cap ORF sequence. Figure 4 NS1, AAV9-TRADE vector genome with a codon-modified splice acceptor. NS2, AAV9-TRADE vector genome with a codon-modified splice donor. NS3, AAV9-TRADE vector genome with a codon-modified splice acceptor and splice donor. PC, positive control using a plasmid template containing the AAV-PHP.B-hSynI-GFP-TRADE vector genome sequence; NC, no-template PCR control.

[0025] Fig.13 Study design using TRADE to identify AAV variants with enhanced brain neuron transduction after systemic AAV vector injection. A is a diagram of the AAV9-N272A-hSynI-GFP-TRADE-PepLib vector genome. The hSynI enhancer-promoter is used to drive specific expression in neurons. Liver off-target AAV9-N272A cap (PCT / US2017 / 068050) as an AAV library generation platform. A random 8-amino acid peptide encoded by (NNK)8 and flanked by a glycine-serine linker (linker) (SEQ ID NO:2) replaced Q588 of the AAV9-N272A cap sequence (SEQ ID NO:222). The plasmid library of B was used to generate an AAV library using a triple transfection protocol. The library was purified by PEG precipitation and two rounds of CsCl ultracentrifugation and then concentrated at 3×10 11 vg / mouse was injected via tail vein. Brain tissue was harvested 12 days after injection. RNA was recovered using TRIzol, and the cap fragment containing the peptide insertion was recovered using RT-PCR, which was then cloned back into the AAV vector plasmid backbone. Three rounds of selection were repeated in C57BL / 6J mice. At the same time, 2.7×10 12 vg / kg in a single round of selection in rhesus monkeys.

[0026] Figures 14A-14D Validation of neuronal transduction by 26 novel AAV capsids in mice and nonhuman primates by AAV RNA Barcode-Seq. Fig.14AMap of the double-stranded (ds) AAV-hSynI-GFP-BC vector. A pair of two 12-nucleotide-long DNA barcodes (VBCx-L and VBCx-R) are placed under the human synapsin I (hSynI) gene enhancer-promoter. These two viral barcodes (VBC) can be specifically expressed as transcripts in cells (i.e., neurons) where the hSynI enhancer-promoter is active. Fig. 14B Neuronal transduction with 26 novel AAV variants HN1 to HN26 identified by TRADE (5 variants identified in mice and 21 variants identified in non-human primates) and 3 control AAV capsids (AAV9, AAV9-N272A, and AAV-PHP.B) in C57BL / 6J and BALB / cJ mice. A DNA / RNA barcoded dsAAV-hSynI-GFP-BC library (dsAAV-hSynI-GFP-BCLib) containing 26 novel AAV variants identified by TRADE (5 variants identified by TRADE in mice and 21 variants identified by TRADE in non-human primates) and control AAV capsids (AAV9, AAV9-N272A, and AAV-PHP.B) was injected at 5×10 per mouse. 11 The dose of vg was intravenously injected into three adult male C57BL / 6J mice and three adult male BALB / cJ mice. Two weeks after injection, various tissues were harvested and analyzed for transduction at the AAV vector genome transcription level by AAVRNA Barcode-Seq. The transduction level is expressed as a phenotypic difference (PD) value relative to the reference control AAV9. The AAV capsid amino acid sequence information of HN1 to HN26 variants is shown in Table 3. Fig. 14C Neuronal transduction of 26 novel AAV variants and 3 control AAV capsids in the hippocampus of rhesus monkeys. The same DNA / RNA barcoded AAV library was transduced at 2×10 13 vg / kg was intravenously injected into a juvenile male rhesus monkey. Two weeks after injection, different brain regions were harvested and analyzed for transduction by AAVRNA Barcode-Seq. Fig.14D In use Fig. 14C The relative neuronal transduction efficiencies of the three TRADE variants HN1, HN2, and HN3 and AAV-PHP.B were analyzed by AAV RNA Barcode-Seq in 12 different brain regions of a single rhesus monkey. Fig. 14B , Fig. 14C and Fig.14D In the figure, the dotted line represents the PD value of AAV9 (i.e., 1.0).

[0027] Fig.15Enhanced neuronal transduction of AAV9-N272A-HN1 in mice was verified using a conventional eGFP reporter vector and histological quantification. We prepared AAV9, AAV-PHP.B, and AAV9-N272A-HN1 vectors containing a self-complementary AAV genome expressing eGFP under the control of the hSynI enhancer-promoter (dsAAV-hSynI-eGFP). The purified vectors were inoculated at 3 × 10 11 The dose of vg / mouse was administered via the tail vein to 8-week-old male C57BL / 6J or BALB / cJ mice (n=4 mice / vehicle / mouse strain). Three weeks after injection, mice were transcardially perfused with 4% paraformaldehyde and brain tissues were processed for immunohistochemistry. Fig.15 A in the figure is a diagram of the self-complementary hSynI-eGFP vector genome. Fig.15 Shown in B are representative tile scan images of sagittal sections stained with anti-GFP antibody. Fig.15 C is quantification of neuronal transduction in B based on automated counting of cells expressing eGFP and NeuN in four brain regions. Fig.15 D is a validation of the automated counting process in B and C. Representative 20× confocal images from the visual cortex are shown. Scale bar = 100 μm. Fig.15 Middle E is quantification of neuronal transduction in D based on manual counting of cells expressing eGFP and NeuN by a blinded observer. Error bars represent mean + / - SEM. ***p<0.001.

[0028] Figure 16A-16B Enhanced AAV9-N272A-HN1 transduction relative to AAV9 in rhesus macaques was validated using an epitope-tagged eGFP reporter vector. Fig.16A AAV-CAG-nlsGFP vectors used in this study. We prepared 4 AAV vectors: AAV9-CAG-FLAGnlsGFP-BCLib, AAV9-CAG-HAnlsGFP-BCLib, AAV9-N272A-HN1-CAG-FLAGnlsGFP-BCLib, and AAV9-N272A-HN1-CAG-HanlsGFP-BCLib. nlsGFP (eGFP with a nuclear localization signal derived from the SV40 large T antigen) was tagged at the N-terminus with a FLAG tag or an HA tag. Each vector was a DNA / RNA barcoded library containing an approximately 1:1 mixture of 9 different DNA / RNA barcoded viral clones; however, this feature was not used in this study. Fig.16AThe two vectors in the upper half depicted in the figure were mixed in a 1:1 ratio to make AAV library 1 (AAVLib1), and the two vectors in the lower half were mixed in a 1:1 ratio to make AAV library 2 (AAVLib2). In this experimental scheme, AAVLib1 and AAVLib2 each contained a 1:1 ratio of an AAV9 vector expressing epitope-tagged nlsGFP and an AAV9-N272A-HN1 vector, but the capsid-epitope relationship was inverted to avoid potential antibody bias in downstream analysis. Fig. 16B Representative large-scale tile-scanned brain sections from one animal that received AAVLib. Each AAV library was expressed at 3 × 10 13 vg / kg was injected intravenously into infant rhesus monkeys. Tissues were harvested 3 weeks after injection, cut into 4 mm thick slices, fixed in 4% paraformaldehyde, and analyzed by immunohistochemistry with anti-GFP, anti-FLAG, and anti-HA antibodies. eGFP expression indicates that cells were transduced by AAV9 or AAV9-N272A-HN1, or both. FLAG staining indicates AAV9 capsid-mediated transduction, while HA staining indicates AAV9-N272A-HN1-mediated transduction. Upper right inset, motor cortex; lower right inset, dura mater. This experiment shows that AAV9-N272A-HN1 transduces brain cells several times better than AAV9, with a strong neuronal tropism. Therefore, in terms of neuronal transduction, AAV9-N272A-HN1 mediates much higher neuronal transduction than AAV9.

[0029] Figures 17A-17D Biodistribution of AAV9-N272A-HN1 to major peripheral organs after systemic delivery in mice and rhesus monkeys. We used AAV DNA Barcode-Seq to determine the distribution of AAV9-N272A-HN1 to major peripheral organs after systemic delivery in mice and rhesus monkeys. Fig.17A , Fig. 17B and Fig. 17C ) in each peripheral organ delivered by each AAV capsid contained in the dsAAV-hSynI-GFP-BCLib library. As explained previously, the dsAAV-hSynI-GFP-BCLib library containing 26 AAV variants identified by TRADE in mice and non-human primates, as well as control AAV9, AAV9-N272A, and AAV-PHP.B DNA (see Table 3) were extracted from various tissues after administration of the dsAAV-hSynI-GFP-BCLib library and subjected to AAV DNA Barcode-Seq analysis. We also used AAVRNA Barcode-Seq to determine Fig.16A ( Fig.17D) compared with AAV9 in each peripheral organ of rhesus monkeys injected intravenously with the ssAAV-CAG-nlsGFP-BCLib library. Fig.17A Biodistribution of AAV9-N272A, AAV-PHP.B, and TRADE variants relative to AAV9 to the liver in C57BL / 6J mice, BALB / cJ mice, and rhesus monkeys. Fig. 17B Biodistribution of AAV9-N272A-HN1 to major peripheral organs other than the liver in C57BL / 6J mice and BALB / cJ mice (n=3 mice / strain). Fig. 17C Biodistribution of AAV9-N272A-HN1 to major peripheral organs other than the liver in rhesus monkeys (n=1) based on dsAAV-hSynI-GFP-BC analysis. Fig.14D AAVDNA Barcode-Seq analysis was performed on samples collected from a rhesus macaque injected with the dsAAV-hSynI-GFP-BCLib library as shown. Fig.17D Biodistribution of AAV9-N272A-HN1 to major peripheral organs other than the liver in rhesus monkeys based on ssAAV-CAG-nlsGFP-BC analysis (n=2). Fig.16A Samples collected from rhesus macaques injected with ssAAV-GAG-nlsGFP-BCLib vector as indicated were subjected to AAV RNA Barcode-Seq analysis. Error bars represent mean + / - SEM. AAV9-N272A-HN1 capsids transduced peripheral organs to a lesser extent than AAV9 capsids.

[0030] Fig.18 AAV9-N272A-HN1 is highly neurotropic after systemic administration in mice. AAV9 and AAV9-N272A-HN1 vectors expressing nlsGFP under the control of the strong ubiquitous CAG promoter were injected at 3 × 10 11 The dose of 1000 mg / ml was injected intravenously into 8-week-old male BALB / cJ mice. Tissues were harvested 12 days after injection and analyzed by immunostaining with anti-GFP and anti-NeuN antibodies. Fig.18 Center A is a diagram of the single-stranded (ss) AAV-CAG-nlsGFP vector genome used in this study. Fig.18 Middle B Representative images from the cerebral cortex of mice transduced with AAV9-N272A-HN1-CAG-nlsGFP. The vast majority of cells transduced with AAV9-N272A-HN1-CAG-nlsGFP were also positive for the neuronal marker NeuN. Scale bar = 100 μm. Fig.18Figure C shows the neuronal specificity of AAV9 and AAV9-N272A-HN1 capsids. Quantification of neuronal specificity was determined by dividing the number of double positive cells (eGFP+ / NeuN+) by the total number of GFP+ cells. AAV9-N272A-HN1 was highly specific for neurons (96%) compared to AAV9 (56%). DETAILED DESCRIPTION

[0031] In some embodiments, the present disclosure provides a TRADE system that allows the directed evolution of AAV capsids using antisense mRNA of cap ORF expressed in a cell type-specific or ubiquitous manner. Such a system does not require Cre transgenic animals. Therefore, it can be applied to the cell type-specific AAV capsid evolution of large animals including non-human primates, for which Cre transgenic strains are not easily available. Any cell type-specific or tissue / organ-specific enhancer / promoter or ubiquitous enhancer / promoter can be easily applied to the system without transgenes. Cell type-specific selection is given at the mRNA level. In certain embodiments, multiple directed evolution schemes can be combined into one directed evolution scheme. For example, the selection of neuron-specific AAV capsids, astrocyte-specific AAV capsids, oligodendrocyte-specific AAV capsids, and microglia-specific AAV capsids based on cell type-specific transgenic mRNA expression can be performed simultaneously in a single animal.

[0032] In some embodiments, the present disclosure provides a sense strand TRADE system that allows the directed evolution of AAV capsids using mRNA of cap ORFs that can express AAV capsid proteins in a cell type-specific or ubiquitous manner in target cells. Sense strand TRADE has the same advantages as the antisense strand TRADE for which data are provided herein, because it does not require Cre transgenic animals, gives cell type-specific selection at the mRNA level, and can combine multiple directed evolution schemes into a directed evolution round completed in a single animal. However, a possible disadvantage is that immunogenic AAV capsid proteins may inevitably continue to be expressed in target cells, which may lead to undesirable results during capsid selection.

[0033] In some embodiments, the present disclosure also provides novel AAV capsids. In certain embodiments, these novel AAV capsids transduce brain neurons several times better than AAV9 after intravenous injection in C57BL / 6J mice. In certain embodiments, the novel AAV capsids transduce up to 8 times better than AAV9 after intravenous injection in C57BL / 6J mice. Neuronal transduction levels can be greatly improved compared to AAV9, although they may not reach the levels obtained with AAVPHP.B. In certain embodiments, the novel AAV capsids can transduce brain neurons more effectively than AAVPHP.B.

[0034] In some embodiments, the present disclosure provides novel AAV capsids that can transduce brain neurons several times better than AAV9 after intravenous injection in BALB / cJ mice. In certain embodiments, the novel AAV capsids can transduce brain neurons up to 7 times better than AAV9 after intravenous injection in BALB / cJ mice. The transduction level is much higher than AAVPHP.B.

[0035] In some embodiments, the present disclosure provides novel AAV capsids that can transduce brain neurons several times better than AAV9 after intravenous injection in rhesus monkeys. In certain embodiments, novel AAV capsids can transduce brain neurons up to 4 times better than AAV9 after intravenous injection in rhesus monkeys. These transduction levels are better than AAVPHP.B.

[0036] In some embodiments, the disclosure provides AAV capsids that can transduce lung cells with neuronal cell marker expression several-fold better than AAV9. In certain embodiments, AAV capsids can transduce such cells up to 17-fold better than AAV9.

[0037] In some embodiments, the novel AAV capsids exhibit a liver off-target phenotype.

[0038] In some embodiments, the present disclosure provides codon-modified AAV cap sequences that are not spliced ​​when expressed in the antisense direction. We observed that unmodified AAV cap ORFs are spliced ​​when expressed in the antisense direction (e.g., AAV1, AAV3, and AAV9). In contrast, some codon-modified AAV cap ORFs described in the present disclosure are not spliced. Based on our understanding of the putative splice donor site and splice acceptor site, it is possible to design such a non-spliced ​​version of the AAV cap ORF. When mutations in the cap gene occur in a wide region of the cap ORF, the use of such a non-spliced ​​cap ORF can be used for directed evolution using the TRADE system.

[0039] As used herein, the term "AAV vector" refers to an AAV vector that contains or is derived from AAV components and is suitable for infecting mammalian cells, including human cells; any of a variety of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen or pancreas, whether in vitro or in vivo. The term "AAV vector" can be used to refer to an AAV-type viral particle (or virion) that contains at least a nucleic acid molecule encoding a protein of interest.

[0040] In addition, the AAV disclosed herein can be derived from various serotypes including combinations of serotypes (e.g., "pseudotyped" AAV), or from various genomes (e.g., single-stranded or self-complementary). In specific embodiments, the AAV vectors disclosed herein can comprise a desired protein or protein variant. As used herein, "variant" refers to an amino acid sequence that is altered by one or more amino acids. A variant can have "conservative" changes, in which the substituted amino acids have similar structural or chemical properties, such as replacing leucine with isoleucine. Alternatively, a variant can have "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor changes can also include amino acid deletions or insertions, or both.

[0041] Provided herein are nucleotide sequences, e.g., polynucleotides, encoding proteins of the present disclosure. The nucleotides of the present disclosure may be composed of RNA or DNA. The present disclosure also includes those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein.

[0042] Due to the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode proteins of the present disclosure. In addition, it is well within the skill of those skilled in the art to generate alternative polynucleotide sequences encoding proteins that are identical or substantially identical to the proteins disclosed herein. These variants or alternative polynucleotide sequences are within the scope of the present disclosure. As used herein, reference to "substantially identical sequences" refers to one or more sequences that encode amino acid substitutions, deletions, additions or insertions that do not eliminate the detectability of the polypeptides encoded by the polynucleotides of the present disclosure.

[0043] The present disclosure also includes variants of the polynucleotides and polypeptides disclosed herein. Variant sequences include those in which one or more peptides or nucleotides of the sequence have been substituted, deleted and / or inserted.

[0044] The polynucleotide and polypeptide sequences of the present disclosure can also be defined according to specific identities and / or similarities with certain polynucleotides and polypeptides described herein. The sequence identity is usually greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identities and / or similarities of the sequences can be 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical compared to the sequences disclosed herein. Unless otherwise indicated, as used herein, the sequence identity and / or similarity percentage of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed using the NBLAST program, score = 100, word length = 12 to obtain sequences with the desired sequence identity percentage. In order to obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al. (1997) can be used. When using BLAST and gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used.

[0045] Methods for preparing AAV vectors as disclosed herein are well known in the art, including, for example, methods using packaging cells, helper viruses or plasmids and / or baculovirus systems. See, for example, Amulski et al., J. Virology 63, 3822 (1989); Xiao et al., J. Virology 72, 2224 (1998); Inoue et al., J. Virology 72, 7024 (1998); WO 1998 / 022607; and WO 2005 / 072364.

[0046] Methods for preparing pseudotyped AAV vectors (see, e.g., WO00 / 28004) are also known, as are various modifications or formulations of AAV vectors to reduce their immunogenicity when administered in vivo (see, e.g., WO01 / 23001; WO00 / 73316; WO04 / 112727; WO05 / 005610; and WO99 / 06562). In some embodiments, AAV vectors can be prepared or derived from various serotypes of AAV, which can be mixed together or mixed with other types of viruses to produce chimeric (e.g., pseudotyped) AAV viruses.

[0047] In certain embodiments, the AAV vector can be a human serotype AAV vector. In such embodiments, the human AAV can be derived from any known serotype, such as from serotypes 1-11, such as from any of AAV1, AAV2, AAV4, AAV6, or AAV9.

[0048] The AAV vector disclosed herein may include a nucleic acid encoding a target protein. In various embodiments, the nucleic acid may also include one or more regulatory sequences that allow the target protein to be expressed and secreted in some embodiments, such as a promoter, an enhancer, a polyadenylation signal, an internal ribosome entry site ("IRES"), a sequence encoding a protein transduction domain ("PTD"), a 2A peptide, etc. Therefore, in some embodiments, the nucleic acid may include a promoter region operably connected to the coding sequence to cause or improve the expression of the target protein in infected cells. For example, such a promoter may be ubiquitous, cell or tissue specific, strong, weak, regulated, chimeric, etc., to allow effective and stable production of proteins in infected tissues. The promoter may be homologous to the encoded protein or heterologous, although the promoter commonly used in the disclosed method is functional in human cells. Examples of regulated promoters include, but are not limited to, promoters containing Tet on / off elements, rapamycin inducible promoters, tamoxifen inducible promoters, and metallothionein promoters. Other promoters that can be used include promoters with tissue specificity for tissues such as kidney, spleen and pancreas. Examples of ubiquitous promoters include viral promoters, particularly CMV promoter, RSV promoter, SV40 promoter, etc., and cellular promoters, such as phosphoglycerate kinase (PGK) promoter and β-actin promoter.

[0049] In some embodiments of the AAV vector disclosed herein, one or more feedback elements can be used to inhibit overexpression of the target protein. For example, some embodiments of the AAV vector can include one or more siRNA sequences targeting exogenous transcripts. In other embodiments, the AAV vector can include one or more other promoters that can be recognized by inhibitory transcription factors. In various embodiments, the AAV vector disclosed herein can include a construct that can generate a feedback loop that can maintain the expression level of the target protein at a physiological level in the body.

[0050] In some embodiments of the AAV vector disclosed herein, the genome editing mechanism can be used to genetically modify the cellular genomic DNA or mRNA transcripts in a site-specific manner. Komor et al., Cell 168, 20-36 (2017); and Katrekar et al., Nature Methods 16: 239-242, 2019. For example, some embodiments of the AAV vector may include CRISPR-related enzymes such as Cas9, DNA base editors, RNA editing enzymes, and / or guide RNAs (gRNAs) to modify nucleic acids in cells in a site-specific manner. In addition, the AAV vector may include a homology repair template (HDR) for genome editing.

[0051] In various embodiments, the AAV vectors disclosed herein may comprise a nucleic acid that may comprise a leader sequence that allows secretion of the encoded protein. In some embodiments, fusion of the transgene of interest with a sequence encoding a secretion signal peptide (usually located at the N-terminus of the secreted polypeptide) may allow production of the therapeutic protein in a form that can be secreted from the transduced cells. Examples of such signal peptides include albumin, β-glucuronidase, alkaline protease, or fibronectin secretion signal peptides.

[0052] As described herein, by non-invasive techniques, effective and long-term expression of therapeutic proteins of interest can be achieved in the brain, heart, lungs, skeletal muscle, kidney, spleen or pancreas by peripheral administration of certain AAV vectors, such as non-AAV9 vectors with AAV9 sequences. This peripheral administration may include any administration route that does not require direct injection into the brain, heart, lungs, skeletal muscle, kidney, spleen or pancreas. More specifically, peripheral administration may include systemic injection, such as intramuscular, intravascular (e.g., intravenous), intraperitoneal, intraarterial or subcutaneous injection. In some embodiments, peripheral administration may also include oral administration (see, e.g., WO96 / 40954), delivery using an implant (see, e.g., WO01 / 91803) or administration by instillation of the respiratory system, such as using a spray, an aerosol or any other suitable formulation.

[0053] In various embodiments, the desired dose of the AAV vector can be adjusted by a skilled person, for example, according to the disease state, the subject, the treatment regimen, etc. In some embodiments, 10 5 Up to 10 12 Viral genomes, such as 10 6 Up to 10 11 , 10 7 Up to 10 11 , or 10 8 Up to 10 11 In other embodiments, exemplary dosages for achieving a therapeutic effect may include at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 Viral titer can also be expressed in transduction units, which can be easily calculated by those skilled in the art.

[0054] In various embodiments, the AAV vectors disclosed herein can be administered in any suitable form, for example, in the form of a liquid solution or suspension, in a solid form suitable for dissolution or suspension in a liquid prior to injection, in a gel form, or in an emulsion form. The vector can be formulated with any suitable pharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc. For example, for injection, a suitable carrier or diluent can be an isotonic solution, a buffer, sterile and pyrogen-free water, or, for example, a sterile and pyrogen-free phosphate buffered saline solution. For inhalation, the vector can be in particulate form.

[0055] The vector can be administered in a "therapeutically effective" amount, e.g., an amount sufficient to alleviate (e.g., reduce, lower) at least one symptom associated with a disease state or provide an improvement in the condition of the subject. In some embodiments, repeated administration can be performed, e.g., using the same or different peripheral administration routes and / or the same vector or different vectors.

[0056] The present invention provides embodiments including but not limited to the following:

[0057] 1. A nucleic acid comprising:

[0058] a Parvoviridae genome flanked by ITR sequences, wherein the Parvoviridae genome comprises a Parvoviridae intron, a Parvoviridae cap gene, and a first polyadenylation signal in a first direction;

[0059] a first promoter in said first direction driving expression of the Parvoviridae cap gene in the presence of adenoviral helper function;

[0060] A second promoter and a second polyadenylation signal in a second orientation that is antisense relative to the first orientation, wherein the second polyadenylation signal is located at a position that causes termination of transcription of the antisense mRNA of the Parvoviridae cap gene.

[0061] 2. The nucleic acid of embodiment 1, wherein the second promoter is a cell type-specific promoter.

[0062] 3. The nucleic acid of embodiment 1, wherein the second promoter is a ubiquitous promoter.

[0063] 4. The nucleic acid of any one of embodiments 1-3, wherein the Parvoviridae genome is an AAV genome comprising an AAV intron and an AAV cap gene.

[0064] 5. The nucleic acid of embodiment 4, wherein the AAV cap gene is a wild-type AAV cap gene.

[0065] 6. The nucleic acid of embodiment 5, wherein the AAV cap gene sequence is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or other natural AAV isolate cap gene sequence.

[0066] 7. The nucleic acid of embodiment 4, wherein the AAV cap gene is an engineered AAV cap gene.

[0067] 8. The nucleic acid of embodiment 4, wherein the AAV cap gene is one of a plurality of AAV cap genes in a library.

[0068] 9. A nucleic acid library comprising a plurality of nucleic acids according to embodiment 4, wherein the nucleic acids comprise a plurality of unique AAV cap gene sequences.

[0069] 10. The nucleic acid library of embodiment 9, wherein the nucleic acid library comprises greater than about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 or 10 8 A unique AAV cap gene sequence.

[0070] 11. The nucleic acid as described in any one of embodiments 1-8, further comprising a target gene in the second direction.

[0071] 12. The nucleic acid of any one of embodiments 4-8 or 11, wherein the second polyadenylation signal is located within the AAV intron.

[0072] 13. A nucleic acid as described in any one of embodiments 4-8 or 11, wherein the second polyadenylation signal is positioned so that the cap gene is correctly translated into a full-length capsid protein in the first direction and the cap gene is correctly transcribed into an antisense mRNA comprising a full-length AAV cap gene coding sequence.

[0073] 14. A method for identifying an AAV vector having a cap gene sequence that has an enhanced ability to transduce cells from a tissue of interest when compared to at least one other AAV vector having a different cap gene sequence, the method comprising:

[0074] Prepare a first-round AAV TRADE vector library by introducing the nucleic acid library described in embodiment 9 or 10 into an AAV packaging cell line and recovering the first-round AAV TRADE vector library from the packaging cell line;

[0075] Inject one or more animals with the first round AAV TRADE vector library;

[0076] Recovering the cap gene sequence of the AAV vector enriched in the cells of the target tissue of the animal;

[0077] preparing a second-round AAV TRADE nucleic acid library comprising recovered cap gene sequences of the enriched AAV vectors, introducing the library into an AAV packaging cell line, and recovering a second-round AAV TRADE vector library from the packaging cell line;

[0078] performing a second round of enrichment by injecting the second round AAV TRADE vector library into one or more animals and recovering the cap gene sequences enriched in cells of the target tissue of the animals; and

[0079] Enriched AAV cap gene sequences are identified after the first round of enrichment, after the second round of enrichment, and after any subsequent rounds of enrichment.

[0080] 15. A method for preparing an AAV TRADE vector or an AAV TRADE vector library, comprising:

[0081] The nucleic acid of any one of embodiments 4-8 or 11-13 or the nucleic acid library of embodiment 9 or 10 is introduced into an AAV packaging cell line and the AAV TRADE vector or the AAV TRADE vector library is recovered from the packaging cell line.

[0082] 16. A method for determining a novel cap gene sequence of an AAV vector having an enhanced ability to transduce cells from a tissue of interest, comprising:

[0083] Identifying the AAV vector according to the method described in embodiment 14;

[0084] Recovering antisense mRNA comprising the cap gene sequence; and

[0085] Determine the novel cap gene sequence.

[0086] 17. A method as described in embodiment 16, wherein the antisense mRNA is recovered using RT-PCR.

[0087] 18. The method described in embodiment 16 or 17 further includes a step of determining the cap gene sequence.

[0088] 19. An AAV vector comprising the nucleic acid of any one of embodiments 4-8 or 11-13.

[0089] 20. A nucleic acid comprising:

[0090] a Parvoviridae genome flanked by ITR sequences, wherein the Parvoviridae genome comprises a Parvoviridae intron, a Parvoviridae cap gene, and a first polyadenylation signal in a first direction;

[0091] a first promoter in said first direction driving expression of said Parvoviridae cap gene in the presence of adenoviral helper function; and

[0092] A second promoter in the first orientation driving expression of the Parvoviridae cap gene in the absence of adenoviral helper functions.

[0093] 21. The nucleic acid of embodiment 20, wherein the second promoter is a cell type-specific promoter.

[0094] 22. The nucleic acid of embodiment 20, wherein the second promoter is a ubiquitous promoter.

[0095] 23. The nucleic acid of any one of embodiments 20-22, wherein the Parvoviridae cap gene is a wild-type AAV cap gene.

[0096] 24. The nucleic acid of embodiment 23, wherein the AAV cap gene sequence is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or other natural AAV isolate cap gene sequence.

[0097] 25. The nucleic acid of any one of embodiments 20-22, wherein the Parvoviridae cap gene is an engineered AAV cap gene.

[0098] 26. The nucleic acid of any one of embodiments 20-25, wherein the Parvoviridae cap gene is one of a plurality of AAV cap genes in a library.

[0099] 27. A nucleic acid library comprising a plurality of nucleic acids of embodiment 20, wherein the nucleic acids comprise a plurality of unique Parvoviridae cap gene sequences.

[0100] 28. The nucleic acid library of embodiment 27, wherein the nucleic acid library comprises greater than about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 or 10 8 A unique AAV cap gene sequence.

[0101] 29. The nucleic acid as described in any one of embodiments 20-26 also comprises a target gene.

[0102] 30. A method for identifying an AAV vector having a cap gene sequence that has an enhanced ability to transduce cells from a tissue of interest when compared to at least one other AAV vector having a different cap gene sequence, the method comprising:

[0103] Preparing the first-round AAV TRADE vector library by introducing the nucleic acid library of embodiment 27 or 28 into an AAV packaging cell line and recovering the first-round AAV TRADE vector library from the packaging cell line;

[0104] injecting one or more animals with the first round AAV TRADE vector library;

[0105] Recovering the cap gene sequence of the AAV vector enriched in the cells of the target tissue of the animal;

[0106] preparing a second-round AAV TRADE nucleic acid library comprising the cap gene sequences of the recovered enriched AAV vectors, introducing the library into an AAV packaging cell line, and recovering the second-round AAV TRADE vector library from the packaging cell line;

[0107] performing a second round of enrichment by injecting the second round AAV TRADE vector library into one or more animals and recovering cap gene sequences enriched in cells of the target tissue of the animal; and

[0108] Enriched AAV cap gene sequences were identified after the first round of enrichment, after the second round of enrichment, and after any subsequent rounds of enrichment.

[0109] 31. A method for preparing an AAV TRADE vector or an AAV TRADE vector library, comprising:

[0110] The nucleic acid of any one of embodiments 23-26 or 29 or the nucleic acid library of embodiments 27 or 28 is introduced into an AAV packaging cell line and the AAV TRADE vector or the AAV TRADE vector library is recovered from the packaging cell line.

[0111] 32. A method for determining a novel cap gene sequence of an AAV vector having an enhanced ability to transduce cells from a tissue of interest, comprising:

[0112] Identifying the AAV vector according to the method described in embodiment 30;

[0113] Recovering sense mRNA comprising the cap gene sequence; and

[0114] Determine the novel cap gene sequence.

[0115] 33. A method as described in embodiment 32, wherein the sense mRNA is recovered using RT-PCR.

[0116] 34. The method described in embodiment 32 or 33 further includes a step of determining the cap gene sequence.

[0117] 35. An AAV vector comprising the nucleic acid of any one of embodiments 23-26 or 29.

[0118] 36. The nucleic acid of any one of embodiments 1-8 or 11-13, further comprising at least one mRNA splicing inhibitory mutation in the second direction.

[0119] 37. A nucleic acid as described in embodiment 36, wherein at least one mRNA splicing suppressor mutation includes a change in one or more nucleotides within ten nucleotides of the splice donor and / or splice acceptor site.

[0120] 38. The nucleic acid of embodiment 36 or 37, wherein the alteration does not alter the amino acid sequence encoded by the AAV cap gene.

[0121] 39. An AAV cap ORF sequence comprising one or more of the following mutations in the exon-intron junction at the splice donor site:

[0122] AAV1 VP1 cap ORF 1009-CTTAC (linker) CAGCA-1018* (SEQ ID NO: 199)

[0123] AAV3 VP1 cap ORF 1006-CTTAC (linker) CAGCA-1015* (SEQ ID NO: 199)

[0124] AAV1 VP1 cap ORF 1228-TTTAC (linker) CTTCA-1237 (SEQ ID NO: 200)

[0125] AAV3 VP1 cap ORF 1237-TATAC (linker) CTTCG-1246 (SEQ ID NO: 201)

[0126] AAV1 VP1 cap ORF 1331-ATTAC (linker) CTGAA-1340 (SEQ ID NO: 202)

[0127] AAV1 VP1 cap ORF 1434-GCTAC (linker) CTGGA-1443 (SEQ ID NO: 203)

[0128] AAV1 VP1 cap ORF 1502-TTTAC (linker) CTGGA-1510 (SEQ ID NO: 204)

[0129] AAV1 VP1 cap ORF 1803-ATTAC (linker) CTGGC-1812 (SEQ ID NO: 205)

[0130] AAV3 VP1 cap ORF 1803-CTTAC (linker) CTGGC-1812 (SEQ ID NO: 206)

[0131] AAV1 VP1 cap ORF 1835-TGTAC (linker) CTGCA-1844 (SEQ ID NO: 207)

[0132] AAV1 VP1 cap ORF 2189-GTTAC (linker) CTTAC-2198 (SEQ ID NO: 208)

[0133] AAV9 VP1 cap ORF 2189-GATAC (linker) CTGAC-2198 (SEQ ID NO: 209)

[0134] AAV1 VP1 cap ORF 2194-CTTAC (linker) CCGTC-2203 (SEQ ID NO: 210)

[0135] AAV3 VP1 cap ORF 2194-CTAC(linker)ACGAA-2203 (SEQ ID NO:211).

[0136] (*Although the nucleotide numbers are different, they are the corresponding nucleotides of the AAV cap ORF in the sequence alignment.)

[0137] 40. An AAV cap ORF sequence comprising one or more of the following mutations in the exon-intron junction at the splice donor site:

[0138] AAV1 VP1 cap ORF 305-AGCGT (linker) CTGCA-314 (SEQ ID NO: 212)

[0139] AAV1 VP1 cap ORF 414-GGCTC (linker) CTGGA-423 (SEQ ID NO: 213)

[0140] AAV3 VP1 cap ORF 414-GGCTC (linker) CTGGA-423 (SEQ ID NO: 213)

[0141] AAV1 VP1 cap ORF 495-GCCCG (linker) CTAAA-504 (SEQ ID NO: 214)

[0142] AAV9 VP1 cap ORF 495-GCCCG (linker) CTAAA-504 (SEQ ID NO: 214)

[0143] AAV3 VP1 cap ORF 1133-TCACC (linker) CTGAA-1142 (SEQ ID NO: 215)

[0144] AAV1 VP1 cap ORF 1181-ACTGC (linker) CTGGA-1190 (SEQ ID NO: 216)

[0145] AAV1 VP1 cap ORF 1331-ATTAC (linker) CTGAA-1340** (SEQ ID NO: 202)

[0146] AAV3 VP1 cap ORF 1328-ACTAC (linker) CTGAA-1337** (SEQ ID NO: 217)

[0147] AAV1 VP1 cap ORF 1464-CGTTT (linker) CTAAA-1473 (SEQ ID NO: 218)

[0148] AAV1 VP1 cap ORF 1653-AAACA (linker) CTGCA-1662 (SEQ ID NO: 219)

[0149] AAV1 VP1 cap ORF 2054-GGGAG (linker) CTGCA-2063 (SEQ ID NO: 220)

[0150] AAV3 VP1 cap ORF 2054-GGGAG (linker) CTACA-2063 (SEQ ID NO: 463)

[0151] (**Although the nucleotide numbers are different, they are the corresponding nucleotides of the AAVcap ORF in the sequence alignment.)

[0152] 41. An AAV cap ORF sequence comprising one or more of the following mutations in an exon-intron junction at a splice donor or splice acceptor site:

[0153] Splice Donor

[0154] AAV1 VP1 cap ORF 1009-CTTAC (linker) CAGCA-1018* (SEQ ID NO: 199)

[0155] AAV3 VP1 cap ORF 1006-CTTAC (linker) CAGCA-1015* (SEQ ID NO: 199)

[0156] AAV1 VP1 cap ORF 1228-TTTAC (linker) CTTCA-1237 (SEQ ID NO: 200)

[0157] AAV3 VP1 cap ORF 1237-TATAC (linker) CTTCG-1246 (SEQ ID NO: 201)

[0158] AAV1 VP1 cap ORF 1331-ATTAC (linker) CTGAA-1340 (SEQ ID NO: 202)

[0159] AAV1 VP1 cap ORF 1434-GCTAC (linker) CTGGA-1443 (SEQ ID NO: 203)

[0160] AAV1 VP1 cap ORF 1502-TTTAC (linker) CTGGA-1510 (SEQ ID NO: 204)

[0161] AAV1 VP1 cap ORF 1803-ATTAC (linker) CTGGC-1812 (SEQ ID NO: 205)

[0162] AAV3 VP1 cap ORF 1803-CTTAC (linker) CTGGC-1812 (SEQ ID NO: 206)

[0163] AAV1 VP1 cap ORF 1835-TGTAC (linker) CTGCA-1844 (SEQ ID NO: 207)

[0164] AAV1 VP1 cap ORF 2189-GTTAC (linker) CTTAC-2198 (SEQ ID NO: 208)

[0165] AAV9 VP1 cap ORF 2189-GATAC (linker) CTGAC-2198 (SEQ ID NO: 209)

[0166] AAV1 VP1 cap ORF 2194-CTTAC (linker) CCGTC-2203 (SEQ ID NO: 210)

[0167] AAV3 VP1 cap ORF 2194-CTCAC (linker) ACGAA-2203 (SEQ ID NO: 211)

[0168] Splice acceptor

[0169] AAV1 VP1 cap ORF 305-AGCGT (linker) CTGCA-314 (SEQ ID NO: 212)

[0170] AAV1 VP1 cap ORF 414-GGCTC (linker) CTGGA-423 (SEQ ID NO: 213)

[0171] AAV3 VP1 cap ORF 414-GGCTC (linker) CTGGA-423 (SEQ ID NO: 213)

[0172] AAV1 VP1 cap ORF 495-GCCCG (linker) CTAAA-504 (SEQ ID NO: 214)

[0173] AAV9 VP1 cap ORF 495-GCCCG (linker) CTAAA-504 (SEQ ID NO: 214)

[0174] AAV3 VP1 cap ORF 1133-TCACC (linker) CTGAA-1142 (SEQ ID NO: 215)

[0175] AAV1 VP1 cap ORF 1181-ACTGC (linker) CTGGA-1190 (SEQ ID NO: 216)

[0176] AAV1 VP1 cap ORF 1331-ATTAC (linker) CTGAA-1340** (SEQ ID NO: 202)

[0177] AAV3 VP1 cap ORF 1328-ACTAC (linker) CTGAA-1337** (SEQ ID NO: 217)

[0178] AAV1 VP1 cap ORF 1464-CGTTT (linker) CTAAA-1473 (SEQ ID NO: 218)

[0179] AAV1 VP1 cap ORF 1653-AAACA (linker) CTGCA-1662 (SEQ ID NO: 219)

[0180] AAV1 VP1 cap ORF 2054-GGGAG (linker) CTGCA-2063 (SEQ ID NO: 220)

[0181] AAV3 VP1 cap ORF 2054-GGGAG (linker) CTACA-2063 (SEQ ID NO: 463).

[0182] Example

[0183] The following examples are for illustration only. Based on this disclosure, those skilled in the art will recognize that variations of these examples and other embodiments of the disclosed subject matter can be implemented without undue experimentation.

[0184] We applied the TRADE system in C57BL / 6J mice and rhesus monkeys to identify novel AAV capsids that efficiently transduce brain neurons after systemic delivery. The TRADE system utilizes plasmid constructs containing overlapping bicistronic AAV genomes flanked by ITR sequences ( Figure 2 A). In the sense orientation, the AAV2 p40 promoter drives the expression of the AAV cap gene to facilitate efficient production of viral particles ( Figure 2 B). In the antisense orientation, a cell type-specific enhancer-promoter (e.g., the human synapsin I (hSynI) enhancer-promoter) drives expression of a transcript encoding GFP and an antisense cap sequence, terminating with a polyadenylation signal (poly A) embedded in an intron present in the AAV2 genome ( Figure 2 B). Using the TRADE construct as a cloning backbone, we generated an AAV library based on the liver off-target AAV9-N272A (PCT / US2017 / 068050) cap gene platform containing random 8-mer peptides with a glycine-serine linker (5'-GGGS; 3'-GGGGS) substituted at position Q588 in the AAV9 capsid. Capsid sequences were recovered from brain tissue by RT-PCR as antisense cap ORF mRNA for in vivo selection in specific cell types (e.g., neurons). This approach ensured that the recovered sequences were derived only from AAV variants that were able to mediate RNA expression in our infected cells of interest. When the hSynI enhancer-promoter was used, only AAV capsid sequences that were able to transduce neurons were rescued, enabling neuron-specific selection of AAV capsids.

[0185] We first tested the ability of the TRADE system to recover AAV cap gene sequences from cell type-specific antisense mRNAs using the AAV-PHP.B-hSynI-GFP-TRADE vector ( Figure 3 The hSynI enhancer-promoter driven GFP expression cassette was incorporated into the AAV vector genome containing the AAV-PHP.B capsid gene in the TRADE configuration ( Figure 3 A). The vector genome was packaged into AAV-PHP.B capsid, and the resulting AAV vector was injected intravenously into two 8-week-old male C57BL / 6J mice ( Figure 3B). Twelve days after injection, brain tissue was harvested. Tissue fixed with 4% paraformaldehyde was analyzed by immunofluorescence microscopy. Unfixed tissue was used for RNA extraction and RT-PCR analysis. We confirmed that eGFP was expressed only in neurons ( Figure 3 C and Figure 3 D), indicating that the antisense mRNA transcribed from the cap gene is expressed in a cell type-specific manner. We efficiently recovered the antisense mRNA of the cap gene by RT-PCR ( Figure 3 E). Sanger sequencing of the splicing sites unique to antisense mRNA confirmed that the RT-PCR product was indeed derived from the antisense mRNA driven by the hSynI enhancer-promoter ( Figure 3 F). In addition, Sanger sequencing confirmed the sequence of the PHP.B peptide insertion ( Figure 3 F). Taken together, these observations establish the ability of the TRADE system to successfully recover AAV cap sequences from hSynI enhancer-promoter driven antisense mRNA expressed in AAV vector-transduced brain neurons.

[0186] With the successful establishment of the TRADE system, we performed two AAV capsid directed evolution experiments; one using 8-week-old male C57BL / 6J mice and the other using an 8-month-old male rhesus monkey. We generated the AAV9-N272A-hSynI-GFP-TRADE-Lib library, which consisted of AAV9-derived mutant capsids with a GGGS(N8)GGGGS (SEQ ID NO:2) peptide insertion at position Q588, where N8 represents a random 8-mer peptide encoded by (NNK)8. For peptide insertion, Q588 was replaced with each peptide sequence. The diversity of the AAV library was at least 10 7 In the mouse directed evolution experiment, we injected 3 × 10 11 For the second round of selection, we used two mice at a dose of 1 × 10 12 , 1×10 11 , 1×10 10 or 1×10 9 For the third round of selection, we used two mice at 1 × 10 11vg was injected with the AAV library. We harvested brain tissues twelve days after the injection and separated them into three regions, namely, the cerebrum, cerebellum, and brainstem. Only brain samples were used for directed evolution experiments. We extracted total RNA from the brain, reverse transcribed the RNA using oligo dT primers, and amplified the peptide region including the flanking regions by a pair of cap gene-specific PCR primers. The RT-PCR product was then used to create the next AAV9-N272A-hSynI-TRADE-Lib plasmid library, which was subsequently used to generate the next AAV9-N272A-hSynI-TRADE-Lib viral library. For the second and third rounds of selection, we packaged the AAV9-N272A-hSynI-TRADE-Lib genome without the GFP ORF. In the nonhuman primate directed evolution experiments, we injected 2.0 × 10 per kg of 12 vg was infused with the AAV9-N272A-hSynI-GFP-TRADE-Lib library. 12 days after injection, whole brains were harvested and sliced ​​using brain molds, processed with RNAlater (Thermo Fisher Scientific), and cryopreserved. Total RNA was then extracted from the following brain regions: frontal cortex, occipital cortical area, cerebellum (Purkinje and granular layers), medulla, pons, frontal cortex, hypothalamus, thalamus, cingulate gyrus, caudate nucleus, dura, hippocampus, and preoptic area. We retrieved peptide sequences by RT-PCR in the same manner as above, except that we performed nested PCR to obtain PCR products sufficient for downstream Illumina and Sanger sequencing procedures. For some samples, we cloned the first PCR product directly into the plasmid backbone without the need for nested PCR for Sanger sequencing. After three rounds of selection in mice (Table 1) and one round of selection in non-human primates, we identified a number of potential transduction-enhancing peptides inserted into the AAV9 capsid (Table 2). We then generated barcoded AAV libraries and used the DNA / RNA Barcode-Seq technology previously developed by the Nakai lab (Adachi et al. Nat Commun 5, 3075 (2014); and PCT / US2017 / 068050) to compare the transduction efficiency, tropism / biodistribution, and pharmacokinetics of 26 selected novel AAV variants after intravenous administration in two commonly used mouse strains (C57BL / 6J and BALB / cJ) and a rhesus monkey (Table 3). As a result, we found that: (1) Some novel variants identified by the TRADE technology, particularly AAV9-N272A-TTNLAKNS (HN1) and AAV9-N272A-QQNGTRPS (HN2), performed up to 8-fold better than AAV9 in the brain of C57BL / 6J mice ( Fig. 14B and Fig. 14C). For HNx designations, see Table 3. (2) As previously reported by Hordeaux et al. (Hordeaux et al. 2018), AAV-PHP.B transduced the brain of BALB / cJ mice only at levels comparable to or lower than AAV9 ( Fig. 14B and Fig. 14C ), demonstrating mouse strain-dependency of the potent neurotropic enhancement for AAV-PHP.B. (3) In contrast, AAV9-N272A-TTNLAKNS (HN1) and AAV9-N272A-QQNGTRPS (HN2) retained potent neuronal transduction in BALB / cJ mice, showing 7-fold better transduction than AAV9 ( Fig. 14B (4) In rhesus macaques, many novel AAV mutants showed enhanced neuronal transduction in certain brain regions, up to 4-fold compared to AAV9, whereas AAV-PHP.B was similar to or inferior to AAV9 in transducing the nonhuman primate brain. In particular, AAV9-N272A-TTNLAKNS(HN1) best transduced the nonhuman primate brain in multiple brain regions ( Fig. 14C and Fig.14D ). (5) All AAV9-N272A derivatives, including HN1, HN2, and HN3, showed varying degrees of liver off-target properties in mice and rhesus monkeys ( Fig.17A (6) In mice, AAV9-N272A-TTNLAKNS (HN1) and AAV9-N272A-QQNGTRPS (HN2) can transduce cells with hSynI enhancer-promoter transcriptional activity in the lung up to 17 times better than AAV9 ( Fig. 17B , Table 4 and Table 6). (7) AAV9-N272A-TTNLAKNS(HN1) showed less spread of the vector genome to peripheral organs than AAV9 ( Fig. 17C and Fig.17D ). The AAV Barcode-Seq data are summarized in Tables 4 to 9. Representative data presented in Tables 4 to 9 are also shown graphically in Figures 14 and 17. Table 1 h Peptide sequences identified by the SynI-TRA1 system using the AAV9-N272N-GGGS (N8) GGGGS library targeting mouse brain neurons

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] The table lists the peptide insertions on AAV9-N272A after each of the three rounds of selection. The numbers in brackets indicate the frequency of each peptide out of a total of 69 peptides identified after three rounds of selection. Peptides without numbers were found only once. The sequence of the peptide region was determined by Sanger sequencing. The actual peptide sequence is a random octapeptide flanked by glycine-serine linkers such that the Q588 position was replaced by GGGS(N8)GGGGS.

[0194] For example,

[0195] -TNHQSA GGGSTTNLAKNSGGGGS AQAQTG-" is represented by TTNLAKNS and

[0196] -TNHQSA GGGSQQNGTRPSGGGGS AQAQTG-” is represented by QQNGTRPS.

[0197] Table 2 Peptide sequences identified by the hSynI-TRADE system using the AAV9-N272A-GGGS(N8)GGGGS library targeting rhesus monkey brain neurons.

[0198]

[0199] The sequence of the peptide region was determined by Illumina sequencing or Sanger sequencing. The actual peptide sequence was a random octapeptide flanked by a glycine-serine linker such that position Q588 was replaced by GGGS(N8)GGGGS. These peptides were recovered from the frontal cortex, occipital cortical region, hypothalamus, and thalamus.

[0200] Table 3 List of 29 AAV capsids contained in the DNA / RNA barcoded dsAAV-hSynI-GFP-BCLib library used for phenotypic determination of each AAV strain.

[0201]

[0202]

[0203] Novel AAV9-hSynI-TRADE derived capsid variants were selected from those identified after three rounds of selection in mice (Table 1) and one round of selection in rhesus monkeys (Table 2). Each recovered AAV variant was assigned an abbreviation, HNx. A dsAAV-hSynI-GFP-BCLib library containing DNA / RNA barcodes was constructed so that each AAV variant packaged a unique dsAAV-hSynI-GFP-BC viral genome expressing an AAV variant-specific RNA barcode. The number of unique AAV barcode clones for each variant (including the key reference variant) is listed in this table.

[0204] Table 4 Brain neuron transduction efficiency and biodistribution of TRADE-identified AAV variants after intravenous administration in C57BL / 6J mice.

[0205]

[0206]

[0207] The DNA / RNA barcoded dsAAV-hSynI-GFP-BC library (dsAAV-hSynI-GFP-BCLib) containing 26 novel AAV variants identified by TRADE and control AAV capsids were cultured at 5 × 10 per mouse. 11 The dose of vg was intravenously injected into 3 C57BL / 6J mice (for the library, see Table 3). Two weeks after injection, various tissues were collected and analyzed, brain transduction was analyzed by AAVRNA Barcode-Seq, and various tissues were analyzed for biodistribution to peripheral organs by AAV DNA Barcode-Seq. All values ​​were normalized using the values ​​of AAV9 (AAV9=1.0).

[0208] Table 5 Pharmacokinetic profile of TRADE-identified AAV variants following intravenous administration in C57BL / 6J mice.

[0209]

[0210]

[0211] For the injection of 1×10 13Blood samples obtained from mice injected with 100 vg / kg of dsAAV-hSynI-GFP-BCLib library with DNA / RNA barcodes were subjected to AAV DNA Barcode-Seq analysis (see Table 3, n=2). All values ​​were normalized to AAV9 (AAV9=1.0). All values ​​were normalized to AAV9 (AAV9=1.0). Table 6 Brain neuron transduction efficiency and biodistribution of AAV variants identified by TRADE after intravenous administration in BALB / cJ mice.

[0212]

[0213] The DNA / RNA barcoded dsAAV-hSynI-GFP-BC library (dsAAV-hSynI-GFP-BCLib) containing 26 novel AAV variants identified by TRADE and control AAV capsids were cultured at 5×10 per mouse. 11 vg doses were injected intravenously into 3 BALB / cJ mice (for libraries, see Table 3). Two weeks after injection, various tissues were collected and analyzed for brain transduction by AAV RNA Barcode-Seq and for biodistribution to peripheral organs by AAV DNA Barcode-Seq. All values ​​were normalized using the values ​​of AAV9 (AAV9 = 1.0). Table 7 Transduction efficiency of hSynI-TRADE-derived AAV variants in different brain regions of a rhesus monkey after intravenous administration as determined by AAVhSynI-RNA Barcode-Seq analysis

[0214]

[0215]

[0216] 2.0×10 13 AAV RNA Barcode-Seq analysis was performed using RNA extracted from different brain regions of a rhesus monkey (n=1) expressing a DNA / RNA barcoded dsAAV-hSynI-GFP-BCLib library under the control of the hSynI enhancer-promoter at 100 vg / kg. All values ​​were normalized using the values ​​of AAV9 (AAV9=1.0).

[0217] Table 8 Pharmacokinetic profile of hSynI-TRADE derived AAV variants after intravenous administration in rhesus monkeys.

[0218]

[0219]

[0220] For the injection of 2×10 13 AAV DNA Barcode-Seq analysis was performed on blood samples obtained from one rhesus monkey that received 100 vg / kg of the DNA / RNA barcoded dsAAV-hSynI-GFP-BCLib library (same animal as in Table 7). All values ​​were normalized to those of AAV9 (AAV9=1.0).

[0221] Table 9 Biodistribution of hSynI-TRADE derived AAV variants to peripheral tissues of rhesus monkeys after intravenous administration as determined by AAV DNA Barcode-Seq analysis

[0222]

[0223]

[0224] 2×10 13 AAV DNA Barcode-Seq analysis was performed on DNA extracted from various peripheral tissues of one rhesus monkey (n=1, the same animal as shown in Table 7) that was loaded with 100 vg / kg of the dsAAV-hSynI-GFP-BCLib library with DNA / RNA barcodes. All values ​​were normalized to AAV9 (AAV9=1.0).

[0225] Table 9 Splice donor and splice acceptor sites identified in antisense AAV cap ORF transcripts.

[0226]

[0227] Ten nucleotides surrounding the exon-intron junction identified in the antisense AAV cap mRNA are present with the junction at the center. Underlined letters represent intron sequences. Letters without underline represent exon sequences. Numbers represent nucleotide positions of the AAVcap ORF. SD, splice donor; SA, splice acceptor. Please note that SEQ ID NO: 199 of AAV1 and SEQ ID NO: 199 of AAV3 correspond to each other in sequence alignment. Similarly, SEQ ID NO: 213 of AAV1 and SEQ ID NO: 213 of AAV3 correspond to each other in sequence alignment.

[0228] During the experiment, when the AAV9 cap gene ORF was expressed in the antisense orientation in HEK293 cells or Neuro2a cells, most of the antisense AAV9 cap gene mRNA-derived RT-PCR products were truncated by approximately 1.7 kb ( Figure 4 ), although this was not the case for RNA recovered from brain tissue of mice transduced with AAV-PHP.B-hSynI-GFP-TRADE ( Figure 3 ). Sequencing of the truncated RT-PCR product showed that a 1694 bp region was missing from the AAV9 cap ORF ( Figure 5 Without being bound by any particular theory, based on the observation that we can identify splice donor and acceptor sites in PCR products with common features of exon-intron junctions, truncations appear to be caused by splicing events. Interestingly, sequence alignment studies have shown that cryptic splice donor and acceptor sites with common features of exon-intron junctions can also be identified in many natural AAV serotypes in regions corresponding to the splice donor and acceptor sites identified in the AAV9 cap gene and are highly conserved ( Figure 6 ). This suggests that splicing may occur in the capORF-derived antisense mRNA of not only AAV9 but also many other AAV strains. To date, we have shown that splicing occurs on the AAV3 cap ORF-derived antisense mRNA when it is expressed under the control of the human liver-specific promoter (LSP) in HepG2 cells. Although full characterization has not yet been completed, preliminary RT-PCR using antisense mRNA-specific RT primers produced truncated RT-PCR products as well as full-length non-spliced ​​products. Sequencing analysis of the two truncated RT-PCR products showed the presence of multiple splicing events on the antisense mRNA ( Figure 7 Sequencing studies identified other potential splice donor and acceptor sites (Figures 8 and Fig. 9 We also found splicing events in antisense mRNAs derived from the AAV1 cap ORF when the antisense mRNAs were transcribed from the hSynI enhancer-promoter in HEK293 cells or Neuro2a cells ( Fig.10 Many of the identified splice donor sites (GT / CA) and splice acceptor sites (AG / TC) are highly conserved across different serotypes, suggesting the possibility that these sites also function as splice donor and splice acceptor sites across AAV serotypes, a possibility that warrants further investigation. In fact, we found that splicing of antisense mRNA transcripts of the AAV1, AAV3, and AAV9 cap ORFs uses several common splice donor and splice acceptor sites ( Fig.10 To date, we have not found splicing of the antisense mRNA transcript of the AAV5 cap ORF. For serotypes other than AAV1, AAV3, AAV5, and AAV9, splicing events in the antisense mRNA of the AAV cap ORF have not been studied.

[0229] Potential splicing of cap ORF-derived antisense mRNAs is scientifically interesting but can be an impediment to the TRADE system when it is desired to recover the full-length cap ORF sequence from the antisense mRNA. To overcome this potential problem, we introduced silent mutations that are likely to disrupt conserved sequences at exon-intron junctions and branch points. To demonstrate the principle of this approach, we introduced silent mutations into the AAV9 cap ORF contained in the plasmid pAAV9-N272A-PHP.B-hSyn1-GFP-TRADE that disrupt the splice acceptor (SA) consensus sequence (pAAV9NS1 construct), the splice donor (SD) consensus sequence (pAAV9NS2 construct), and the splice acceptor and donor consensus sequence (pAAV9NS3 construct). Note that the NS strand stands for “non-spliced.” The methods we used to disrupt these consensus sequences are described below.

[0230] We codon-optimized the AAV cap ORF sequence for expression in human cells.

[0231] To identify potential splice donor and acceptor sites on antisense mRNAs derived from the cap ORF, we based our experimental and bioinformatic observations (i.e., Figure 5 , Figure 6 , Figure 7 Figure 8 Fig. 9 and Fig.10 ) developed and used our proprietary database of potential splice donor and splice acceptor sites on antisense mRNAs.

[0232] We disrupted the GT (splice donor) and / or AG (splice acceptor) consensus sequences by changing at least one nucleotide with a codon-optimized sequence. If the codon-optimized sequence was not applicable, we used alternative nucleotides that could disrupt the consensus sequence.

[0233] We removed a stretch of Ts upstream of the splice acceptor site by introducing silent mutations based on the codon-optimized sequence. If the codon-optimized sequence was not sufficient to disrupt a stretch of Ts, we used alternative nucleotides.

[0234] We also avoided Gs at the ends of exons whenever possible.

[0235] Using several programs that can predict branch points (e.g., Human Splicing Finder (Desmet, Hamroun et al. 2009)), we identified potential branch points and replaced them with codon-optimized sequences. If the degree of nucleotide variation obtainable by this method was insufficient, we introduced alternative nucleotides to disrupt potential branch points.

[0236] Using this approach, we created AAV9NS1 (SA, disrupted), AAV9NS2 (SD, disrupted), and AAV9NS3 (SD and SA, disrupted) cap ORFs ( Fig. 9 We expressed these ORFs in the antisense orientation under the control of the hSynI enhancer-promoter in Neuro2a cells by transient plasmid transfection and analyzed the antisense transcripts by RT-PCR. This experiment showed that splicing could be effectively inhibited in all NS1, NS2, and NS3 cap ORFs ( Fig.10 ). It should be noted that even if splicing occurs on the cap ORF-derived antisense mRNA, it is still possible to recover the relatively small peptide insert region of the cap ORF from the pre-mRNA by RT-PCR.

[0237] The TRADE method described herein uses antisense mRNA for viral protein evolution to establish a proof of principle and show successful simplification of the method to practice. The TRADE system can also utilize mRNA in the sense orientation as long as viruses can be generated and potential expression of viral proteins in target cells during directed evolution does not preclude successful evolution of novel capsids.

[0238] Additional information related to nucleic acid splicing and AAV can be found in Desmet et al., Nucleic Acids Res 37, e67 (2009); Matsuzaki et al., Neurosci Lett 665, 182-188 (2018); and Hordeaux et al., Mol Ther 26, 664-668 (2018).

[0239] All references cited in this disclosure are incorporated by reference in their entirety.

Claims

1. An adeno-associated virus serotype 9 (AAV9) capsid protein comprising a peptide insertion, wherein the peptide insertion comprises the amino acid sequence of SEQ ID NO:

128.

2. An adeno-associated virus serotype 9 (AAV9) capsid protein comprising a peptide insertion, wherein the peptide insertion is any one of the peptide insertion site SEQ ID NOs listed in Table 3.

3. A nucleic acid molecule encoding an adeno-associated virus serotype 9 (AAV9) capsid protein comprising a peptide insertion, wherein the peptide insertion comprises the amino acid sequence of SEQ ID NO:

128.

4. A nucleic acid molecule encoding an adeno-associated virus serotype 9 (AAV9) capsid protein comprising a peptide insertion, wherein the peptide is inserted into any one of the peptide insertion sites SEQ ID NOs listed in Table 3.

5. An adeno-associated virus serotype 9 (AAV9) capsid protein having 99% or higher sequence identity with a variant AAV9 capsid protein, wherein the variant AAV9 capsid protein comprises SEQ ID NO:221 and has a substitution of SEQ ID NO:2 at position 588, wherein X1-X8 of SEQ ID NO:2 is selected from any one of the peptide insertion site SEQ ID NOs listed in Table 3.

6. An adeno-associated virus serotype 9 (AAV9) capsid protein having 99% or higher sequence identity with a variant AAV9 capsid protein, wherein the variant AAV9 capsid protein comprises SEQ ID NO:221 and has a substitution of SEQ ID NO:2 at position 588, wherein X1-X8 of SEQ ID NO:2 is SEQ ID NO:

128.

7. A nucleic acid molecule comprising an adeno-associated virus (AAV) cap gene sequence in an antisense orientation, wherein the AAV cap gene sequence in the antisense orientation comprises a messenger RNA (mRNA) splicing inhibitory mutation.

8. A nucleic acid molecule comprising: (a) a first nucleic acid sequence comprising a first regulatory sequence and an adeno-associated virus (AAV) cap gene sequence in a sense orientation; (b) a second nucleic acid sequence comprising a second regulatory sequence and an AAV cap gene sequence in an antisense orientation, wherein the AAV cap gene sequence in an antisense orientation comprises a messenger RNA (mRNA) splicing inhibitory mutation.

9. A method for isolating a nucleic acid molecule comprising an adeno-associated virus (AAV) cap gene sequence, the method comprising: (a) transducing a cell with an AAV vector comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises the AAV cap gene sequence in an antisense orientation, and the AAV cap gene sequence in the antisense orientation comprises a messenger RNA (mRNA) splicing inhibitory mutation; as well as (b) recovering the antisense AAV cap gene sequence or its transcribed sequence from the cell.

10. An adeno-associated virus (AAV) vector comprising a nucleic acid molecule and an AAV virus particle, wherein: The nucleic acid molecule comprises an AAV cap gene sequence in an antisense orientation, The antisense AAV cap gene sequence comprises a messenger RNA (mRNA) splicing inhibitory mutation; and The AAV viral particle comprises a capsid encoded by the AAV cap gene sequence.

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