Alternative barcoding method to assess transduction efficiency of AAV vectors

Abstract

The invention relates to a barcoding method to assess transduction efficiency of AAV vectors, in particular in vivo in rodent and non-human primate.

Description

[0001] ALTERNATIVE BARCODING METHOD TO ASSESS TRANSDUCTION EFFICIENCY OF AAV VECTORS

[0002] FIELD OF THE INVENTION

[0003] The invention pertains to the field of AAV vectors for gene therapy. The invention relates to a barcoding method for assessing the transduction efficiency of AAV vectors, in particular in vivo in rodent and non-human primate.

[0004] BACKGROUND OF THE INVENTION

[0005] Adeno-associated virus (AAV) vectors are among the most efficient tools for in vivo gene transfer and have already shown their therapeutic potentials to treat many monogenic disorders in several clinical trials.

[0006] AAV is a non-pathogenic virus belonging to the genus Dependoparvovirus within the family Parvoviridae. AAV is a non-enveloped virus composed of a capsid of about 26 nm in diameter and a single-stranded DNA genome of 4.7 kb. The genome carries two genes, rep and cap, flanked by two palindromic regions named Inverted terminal Repeats (ITR) that serve as the viral origins of replication and the packaging signal. The cap gene codes for three structural proteins VP1, VP2 and VP3 that compose the icosahedral AAV capsid through alternative splicing and translation from different start codons. The rep gene encodes four proteins required for viral replication Rep78, Rep68, Rep52 and Rep40. Recombinant AAV vectors (i.e. AAV vector particles or AAV particles) encapsidate an ITR-flanked rAAV genome in which a therapeutic transgene expression cassette replaces the AAV protein coding-sequences.

[0007] However, the lack of efficiency to reach some specific tissues and get transgene expression at therapeutic levels, as well as de-targeting non-essential tissues, remain to date, the key challenges to overcome in order to increase potency and safety of AAV vector.

[0008] Currently, many laboratories are focusing their research on capsid engineering aiming to improve the specificity and efficiency of tissue targeting, de-targeting unwanted tissues and decrease immune response. Engineering methods are often based on the generation of millions of capsids variants followed by rounds of selections, consequently, they require library approaches and subsequent next generation sequencing to be characterized. Moreover, since novel capsids are usually screened in mouse, investigating their efficiency in large animal model close to human is essential.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors have developed a barcoded method to assess the in vivo transduction efficiency of new engineered capsids in mouse and non-human primate (NHP). The method uses unique entirely defined barcode DNA sequences of around 80bp, that were named QRcode. The analysis of biodistribution and transgene expression in different tissues confirmed the reliability and the robustness of the method for the characterization of new AAV vectors. In contrast with classical barcoded library approaches, the QRcode method allows to assess the vector biodistribution of selected AAV variants at medium-throughput by classical qPCR or droplet digital PCR and to deep characterize vectors localization in the tissue by in situ hybridization. The method is also suitable for assessing in vitro transduction efficiency of new engineered capsids.

[0011] The invention relates to a method of preparation of a set of barcoded AAV vectors for assessing AAV vector transduction efficiency, in particular in vivo transduction efficiency, comprising the steps of: a) Providing n different AAV capsids, an AAV vector genome comprising a transgene expression cassette with a polyadenylation signal, and n different nucleic acid barcode sequences of at least 60 nucleotides, wherein each barcode sequence is detectable by in situ hybridization and quantitative polymerase chain reaction using complementary oligonucleotide probe(s); b) Inserting each nucleic acid barcode sequence, separately, in the AAV vector genome between the transgene stop codon and the polyadenylation signal; thereby generating n different barcoded AAV vector genomes that express a transgene transcript comprising one of the barcode sequences and a polyadenylation tail; and c) Encapsidating each barcoded AAV vector genome, separately with one of the AAV capsids to generate different barcoded AAV vector particles which are mixed to generate a set of n barcoded AAV vector particles. In some embodiments, the nucleic acid barcode sequence is a DNA sequence of 60 to 100 nucleotides; preferably of about 80 nucleotides. In some preferred embodiments, the DNA barcode sequence is selected from the group consisting of SEQ ID NO: 1 to 7.

[0012] In some embodiments, the barcode transcript sequence is detectable by both in situ- hybridization and quantitative reverse transcription polymerase reaction and the barcode DNA sequence is detectable by quantitative reverse transcription polymerase reaction.

[0013] In some embodiments, the AAV capsids are from variant AAV serotypes, hybrid AAV serotypes, peptide-modified AAV serotypes, or combination thereof.

[0014] In some embodiments, n is from 2 to 10.

[0015] The invention also relates to a barcoded AAV vector or a set of barcoded AAV vectors obtained by the method of preparation of barcoded AAV vectors according to the present disclosure.

[0016] The invention also relates to a method for assessing AAV vector transduction efficiency, comprising: a) Contacting cells with a set of n barcoded AAV vector particles obtained by the method of preparation of barcoded AAV vectors according to the present disclosure; and b) Detecting the n barcode DNA and transcript sequences in the transduced cells obtained in step a) by quantitative polymerase chain reaction (PCR) and in situ hybridization using complementary oligonucleotide probes; and determining therefrom the AAV vector genome copy number and the transgene expression level and in situ detecting AAV vector transduced cells, for each one of the n barcoded AAV vectors.

[0017] In some embodiments, the method for assessing AAV vector transduction efficiency in vivo, comprises: a) In vivo administration of a set n barcoded AAV vector particles obtained by the method of preparation of barcoded AAV vectors according to the present disclosure to at least one animal; and b) Detecting the n barcode DNA and transcript sequences in tissue sample(s) of the administered animal by quantitative polymerase chain reaction (PCR) and in situ hybridization using complementary oligonucleotide probes; and determining therefrom the AAV vector genome copy number and the transgene expression level and in situ detecting AAV vector transduced cells, for each one of the n barcoded AAV vectors.

[0018] In some embodiments, the oligonucleotide probes are selected from the group consisting of SEQ ID NO: 31 to 37. In some embodiments, the method uses complementary oligonucleotide primers; preferably selected from the following pairs: SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO: 19 and 20; SEQ ID NO: 21 and 22; and SEQ ID NO: 23 and 24.

[0019] In some embodiments, the AAV vector genome copy number is determined by quantitative real-time PCR (qPCR) or Droplet Digital PCR (ddPCR); the transgene transcript level is determined by real-time quantitative reverse transcription PCR (RT-qPCR) or reverse transcription Droplet Digital PCR (RT-ddPCR); and the in situ detection of AAV vector transduced cells is performed by in situ hybridization with signal amplification; preferably using SABER-FISH, USeqFISH, Zombie, RNAscope or BaseScope; preferably BaseScope.

[0020] In some embodiments, the animal is a laboratory rodent such as mouse, or a non-human primate.

[0021] In some embodiments, the analyzed tissues in step (b) include one or more of: liver; heart; skeletal muscles; brain; spinal cord; dorsal root ganglions; spleen; and kidney.

[0022] Another aspect of the invention relates to a kit for the preparation of a set of barcoded AAV vectors, comprising at least n nucleic acid barcode sequences according to the present disclosure; preferably selected from the group consisting of SEQ ID NO: 1 to 7.

[0023] Yet another aspect of the invention relates to a kit for assessing AAV vector transduction efficiency, in particular in vivo, comprising at least a set of oligonucleotide probes complementary to the barcode sequences according to the present disclosure; preferably wherein the oligonucleotide probes are selected from the group consisting of SEQ ID NO: 31 to 37. In some embodiments, the kit further comprises complementary oligonucleotide primers; preferably selected from the following pairs: SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO: 19 and 20; SEQ ID NO: 21 and 22; and SEQ ID NO: 23 and 24.

[0024] The invention further relates to a nucleic acid barcode or a set of nucleic acid barcodes according to the present disclosure; preferably selected from the group consisting of SEQ ID NO: 1 to 7.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The invention relates to a barcoding method for assessing the transduction efficiency of AAV vectors, in particular in vivo transduction efficiency of AAV vectors, particularly in rodent and non-human primate. The invention encompasses nucleic acid barcode sequences and their use in methods of making barcoded AAV vectors. The invention also encompasses the barcoded AAV vectors obtained by the methods according to the present disclosure and their use in methods for assessing transduction efficiency of AAV vectors, in particular in vivo transduction efficiency of AAV vectors. The invention further encompasses kits for implementing the methods according to the present disclosure.

[0027] The nucleic acid barcode sequences according to the invention are useful for assessing the transduction efficiency of a set of n AAV vectors in cells that are transduced simultaneously as a mixture or pool of the n different AAV vectors. In particular, the nucleic acid barcode sequences according to the invention are useful for assessing in vivo transduction efficiency of a set of n AAV vectors that are administered simultaneously (as a mixture or pool of the n different AAV vectors) to one animal. They comprise hybridization region(s) for unique complementary probes which allow the combined detection of vector genome copy number, transgene expression level and in situ detection of AAV vector transduced cell for the different AAV vectors of the mixture, in the same cell samples, in particular tissue samples of the animal administered with the AAV vector.

[0028] Definitions

[0029] “AAV vector” has the standard meaning in the art and relates to a recombinant AAV vector particle composed of an AAV capsid packaging a transgene of interest. Therefore, “AAV vector”, “recombinant AAV vector”, “rAAV”, “AAV or rAAV particle, viral particle or virion” are used interchangeably herein to designate a recombinant AAV vector particle. The nucleic acid part of the AAV vector comprising the transgene is the AAV vector genome, also named recombinant AAV vector genome, “rAAV genome” or “vector genome”. The rAAV genome comprises two Inverted Terminal Repeats (ITRs) flanking the transgene of interest, named 5’-ITR and 3’-ITR which are the only sequences of the AAV genome that are required in cis to produce AAV particles.

[0030] As used herein, a “barcode sequence” is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule (nucleic acid or protein) and utilized as a label in order to identify the target biological molecule of interest. A nucleic acid barcode sequence is a synthetic or heterologous nucleic acid that is incorporated within or appended to a target nucleic acid and utilized as a label in order to identify the target nucleic acid.

[0031] “Transduction efficiency”: The transduction efficiency represents how effectively the AAV vector can deliver and express the desired transgene in target cells, tissue or organ.

[0032] “Tropism or targeting”: Tropism or targeting refers to the preference for specific cell types or tissues. Different AAV vectors may exhibit distinct tropism profiles based in part on different surface properties of AAV capsid conferred by the VR sequences, allowing the interaction with specific receptors of host cells. The biodistribution refers to the whole-body tropism profile at the tissue or organ level after in vivo administration of the AAV vector.

[0033] AAV transduction efficiency or tropism may be determined in vitro or in vivo by measuring the vector copy number (VCN) per cell and / or the expression level of the transgene of interest at the mRNA level according to standard methods that are well-known in the art. The transgene of interest may be a therapeutic gene or a reporter gene. AAV transduction efficiency or tropism may be determined in a target or non-target cell, tissue or organ.

[0034] “Target”: The target refers to the cell, tissue or organ in which transduction with the AAV vector and expression of the gene of interest is desired. The non-target refers to the cell, tissue or organ in which transduction with the AAV vector and / or expression of the gene of interest is avoided (detargeting). The target may be without limitation: muscle including heart and skeletal muscles; nervous system, including brain, spinal cord and dorsal root ganglions; liver; spleen and kidney cell or tissue or a combination of said cells or tissues, or other cell or tissue. The non-target may be liver when the targeting of another cell or tissue such as muscle, nervous system, or other cell or issue as disclosed herein is desired.

[0035] As used herein, the term “muscle” refers to cardiac muscle (i.e. heart), diaphragm and skeletal muscle. The term “muscle cells” refers to myocytes, myotubes, myoblasts, and / or satellite cells. In some embodiments, the target is muscle cell or tissue.

[0036] As used herein, the term “nervous system “, refers to both the central (CNS) and peripheral (PNS) nervous system. The term “central nervous system or CNS” refers to the brain, spinal cord, retina, cochlea, optic nerve, and / or olfactory nerves and epithelium. As used herein, the term CNS cells refer to any cells of the CNS including neurons and glial cells (oligodendrocytes, astrocytes, ependymal cells, microglia). The PNS refers to the nerves and ganglia outside the brain and spinal cord.

[0037] The liver consists of several cell types classified into hepatocytes (liver parenchymal cells) that constitute about 80% of liver cells and non-parenchymal cells represented by endothelial cells, Kupffer cells (resident liver macrophages), fat-storing cells (stellate cells or Ito cells), and pit cells (natural killer cells). Kupffer and endothelial cells form the hepatic reticuloendothelial system and constitute the majority of liver non-parenchymal cell types. As used herein “liver cell” includes hepatocyte. As used herein, “hepatocyte” includes primary hepatocyte such as from adult or fetal liver; hepatocyte matured in vitro, hepatocyte cell line; hepatic progenitor or pluripotent stem cell such as induced pluripotent stem cell (iPS cell), embryonic stem cells, fetal stem cell and adult stem.

[0038] As used herein, the term “animal” refers to a mammal, in particular a laboratory rodent or a non-human primate.

[0039] “a”, “an”, and “the” include plural referents, unless the context clearly indicates otherwise. As such, the term “a” (or “an”), “one or more” or “at least one” can be used interchangeably herein; unless specified otherwise, “or” means “and / or”.

[0040] Nucleic acid barcode

[0041] The nucleic acid barcode sequence comprises a unique sequence of at least 60 nucleotides. Each nucleotide of the barcode sequence may be independently a, g, c or t. Each unique barcode sequence comprises hybridization region(s) for complementary oligonucleotide probe(s) to detect the barcode sequence by both in situ hybridization and quantitative polymerase chain reaction, in particular to detect the barcode transcript (mRNA) sequence by both in .s / 7z / -hybridization and quantitative reverse transcription polymerase reaction and to detect the barcode DNA sequence by quantitative polymerase reaction. The barcode sequence may further comprise hybridization region(s) for the complementary oligonucleotide primers used for the polymerase chain reaction. The detection of the barcode DNA and transcript sequences by both in situ hybridization and quantitative polymerase chain reaction using complementary oligonucleotide probe(s) allows for the combined determination of AAV vector genome copy number, AAV vector transgene expression level and in situ detection of AAV vector transduced cell in cells transduced in vitro or tissue samples transduced in vivo with AAV vectors. The complementary probes and the primers, in particular complementary primers, are designed based on the known sequence of the Barcode, using appropriate software that is well-known in the art. The vector copy genome copy number (VCN) is determined on a genomic DNA extract of AAV vector transduced cells or tissue. The transgene expression level is determined on a total RNA extract of AAV vector transduced cells or tissue. The barcode sequence comprises a single continuous stretch of nucleotides inserted into an untranslated region (UTR), (e.g., the 5’ or 3’ UTR) of an AAV vector genome comprising a transgene expression cassette. The barcode sequence is thus inserted into an untranslated region (UTR), (e.g., the 5’ or 3’ UTR) of the nucleic acid sequence encoding the transgene of interest, thereby allowing the detection of the AAV vector genome copy number, transgene expression level and in situ detection of vector transduced cell by detection of the barcode sequence.

[0042] In some particular embodiments, each unique barcode sequence has no match with rodent, in particular mouse, and non-human primate genomic DNA.

[0043] In some particular embodiments, the nucleic acid barcode sequence has a length of 60 to 100 nucleotides; preferably of about 80 nucleotides.

[0044] In some particular embodiments, the barcode sequence comprises at least one of the following features: a length of 60 to 100 nucleotides; a GC content of less than 50 %; and an intramolecular structure stability, AG superior or equal to -27 kcal mol’1; preferably AG is superior or equal to -15 kcal mol’1. In some more particular embodiments, the barcode sequence comprises at least two of the features, preferably all of the features. In some more particular embodiments, the barcode sequence comprises at least a length of 60 to 100 nucleotides, preferably of about 80 nucleotides.

[0045] The Gibbs free energy change (AG) is the energy required to break the secondary structures, and larger negative values indicate stable, undesirable hairpins that can adversely affect the reaction (PCR or in situ hybridization).

[0046] In some particular embodiments, the nucleic acid barcode sequence is a DNA barcode sequence.

[0047] In some preferred embodiments, the nucleic acid barcode sequence is a DNA barcode sequence selected from the group consisting of SEQ ID NO: 1 to 7.

[0048] In some particular embodiments, the barcode sequence is inserted between the transgene stop codon and the polyadenylation signal; thereby generating a barcoded AAV vector genome that expresses a transgene transcript comprising the barcode sequence and a polyadenylation tail.

[0049] AAV vector genome

[0050] The AAV vector genome is a DNA molecule comprising the transgene flanked by ITRs. The AAV vector genome usually comprises a nucleic acid construct containing a transgene expression cassette flanked by ITRs. As used herein the term “inverted terminal repeat (ITR)” refers to a nucleotide sequence located at the 5 ’-end (5TTR) and a nucleotide sequence located at the 3 ’-end (3 ’ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into the host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are the only viral sequences required in cis for the vector genome replication and its packaging into the viral particles. All other viral genes required for viral vector replication can be provided in trans within the virus-producing cells (packaging cells) as described below. In some embodiments, the ITRs are from AAV. AAV ITRs may have wild-type sequence of any AAV serotype or a variant thereof comprising one or more nucleotide insertion, deletion or substitution. In some particular embodiments, 5’ITR and 3’ITR derived from AAV 2 serotype (AAV2). The AAV vector may be a pseudotyped vector, i.e. its genome and capsid are derived from AAVs of different serotypes. The genome of the rAAV vector may either be a singlestranded or self-complementary double-stranded genome (McCarty et al, Gene Therapy, 2003, Dec., 10(26), 2112-2118). Self-complementary vectors are generated by deleting the terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicating genome is half the length of the wild-type AAV genome have the tendency to package DNA dimers.

[0051] According to the present invention, a "transgene of interest" or “gene of interest” refers to a polynucleotide sequence that encodes a RNA or protein product and that may be introduced into a cell for a sought purpose and is capable of being expressed in said cell. A transgene of interest is a gene useful for a particular application, such as with no limitation, diagnosis, reporting, modifying, therapy and genome editing. For example, the gene of interest may be a therapeutic gene, a reporter gene or a genome-editing enzyme.

[0052] A therapeutic transgene is selected and used to lead to a desired therapeutic outcome, in particular for achieving expression of said therapeutic transgene into a cell, tissue or organ into which expression of said therapeutic transgene is needed (i.e., target cell, tissue or organ). Therapy may be achieved by a number of ways, including by expressing a protein into a cell that does not express said protein, by expressing a protein into a cell that expresses a mutated version of the protein, by expressing a protein that is toxic to the target cell into which it is expressed (strategy used, for example, for killing unwanted cells such as cancer cells), by expressing a new protein into a target cell, by expressing an antisense RNA to induce gene repression, exon skipping or to increase the synthesis of the protein of interest, by expressing a silencing RNA such as a shRNA whose purpose is to suppress the expression of a protein, or by expressing a genome-editing enzyme whose purpose is to modify the expression, sequence or regulation a target gene or cellular pathway.

[0053] The gene of interest is any nucleic acid sequence capable of modifying a target gene or target cellular pathway, in target cells, tissue or organ. For example, the gene may modify the expression, sequence or regulation of the target gene or cellular pathway. In some embodiments, the gene of interest is a functional version of a gene or a fragment thereof. The functional version of said gene includes the wild-type gene, a variant gene such as variants belonging to the same family, codon-optimized versions of said wild-type or variant gene and others, or a truncated version, which preserves the functionality of the encoded protein at least partially. A functional version of a gene is useful for gene replacement therapy to replace a gene, which is deficient or non-functional in a patient or to express a new protein in gene addition therapy. In other embodiments, the gene of interest is a gene which inactivates a dominant allele causing an autosomal dominant genetic disease. A fragment of a gene is useful as recombination template for use in combination with a genome editing enzyme.

[0054] The protein encoded by the transgene of interest is any protein or peptide of interest such as with no limitations a protein encoded by a functional version of a non-functional or deficient gene for gene replacement therapy; an antibody or antibody fragment, a genome-editing enzyme, or another protein for gene addition therapy.

[0055] The RNA encoded by the transgene of interest is advantageously complementary to a target DNA or RNA sequence or binds to a target protein. For example, the RNA is an interfering RNA such as a short interfering or silencing RNA (siRNA), a short hairpin RNA (shRNA), a micro RNA (miRNA), a guide RNA (gRNA) for use in combination with a Cas enzyme or similar enzyme for genome editing, an antisense RNA capable of exon skipping such as a modified small nuclear RNA (snRNA) or a long non-coding RNA. A siRNA, a miRNA or a shRNA may be used to regulate the expression of a target gene having altered expression in a target cell, tissue or organ. The guide RNA in complex with a Cas enzyme or similar enzyme for genome editing may be used to modify the sequence of a target gene, in particular to correct the sequence of a mutated / deficient gene or to modify the expression of a target gene having altered expression in a target cell, tissue or organ. The antisense RNA capable of exon skipping is used in particular to correct a reading frame and restore expression of a deficient gene having a disrupted reading frame. The long non-coding RNA is in particular an antisense long non-coding RNA that activates translation, also named SINEUP (For review see for example Zucchelli et al., RNA biology, 2015, 12, 771-779). In some embodiments, the RNA is a therapeutic RNA.

[0056] A reporter gene according to the present invention is in particular suitable for detection of in vitro or in vivo gene expression, in particular in vivo gene expression. Non-limiting examples of reporter genes include genes encoding luminescent proteins, in particular chemiluminescent proteins such as luciferase and others and fluorescent proteins such as Cyan fluorescent proteins, Green fluorescent proteins (GFP), Yellow fluorescent proteins (YFP), Orange fluorescent proteins, Red fluorescent proteins and others; and genes encoding enzymes and / or secreted proteins such as alkaline phosphate and others. One particular example of reporter gene is secreted embryonic alkaline phosphatase (SEAP) gene which encodes a truncated form of human placental alkaline phosphatase.

[0057] The transgene of interest is a functional gene able to produce the encoded protein, peptide or RNAin target cells, tissue or organ. In some embodiments, the transgene of interest is a human gene. In some embodiments, the sequence of the transgene of interest is optimized for expression in the treated individual, preferably a human individual. Sequence optimization may include a number of changes in a nucleic acid sequence, including codon optimization, increase of GC content, decrease of the number of CpG islands, decrease of the number of alternative open reading frames (ARFs) and / or decrease of the number of splice donor and splice acceptor sites. Sequence optimization may also include reduction of sequence length. The transgene may comprise a shortened sequence to facilitate transgene cloning in rAAV vector or improve transgene expression in target cells or tissue.

[0058] The transgene of interest may encode a protein that remains in the target tissue after synthesis. Alternatively, the transgene may encode a protein that is secreted in the bloodstream after synthesis. To express proteins that are secreted in the bloodstream, the transgene advantageously comprises a signal peptide or signal sequence at the 5 ’end of the coding sequence. Signal peptides (SP) are short peptide sequences which are present at the N- terminus of secretory proteins and are used to target proteins for secretion. Multiple signal peptides are known in the art and publicly available (see in particular, Signal Peptide Website and SPdb sequence databases; Puzzo et al., Sci. Transl. Med., 2017, 9(418): doi: 10.1126). Proteins that are secreted in the bloodstream may be expressed in the form of fusion proteins, wherein the protein of interest is linked to a protein stabilizing moiety or to a target cell receptor binding, to target the secreted protein from the bloodstream to a target organ.

[0059] The transgene of interest is operably linked to appropriate regulatory sequences for expression of a transgene in the individual’s target cells, tissue(s) or organ(s). The term “operably linked” as used herein refers to the arrangement of various nucleic acid elements such that the elements are functionally connected and are able to interact with each other. The regulatory sequences which are well-known in the art include in particular a promoter, and further regulatory sequences capable of further controlling the expression of a transgene, by decreasing or suppressing its expression in certain tissues that are not of interest, of by stabilizing the mRNA encoded by the transgene of interest, i.e., coding for the protein or RNA of interest. These sequences include without limitation, silencer, in particular tissue-specific silencer, particularly microRNA target sequence; intron; enhancer; transcription termination signal (polyadenylation signal), and post-transcriptional regulatory element, such as the Woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE). The transgene gene of interest is operably linked to a ubiquitous, tissue-specific or inducible promoter which is functional in cells of target organs. The transgene of interest may be inserted into an expression cassette further comprising additional regulatory sequences as disclosed above. Examples of ubiquitous promoters include the chimeric CAG promoter (comprising the cytomegalovirus early enhancer element, the first exon and the first intron of chicken beta-actin gene and the splice acceptor of the rabbit beta-globin gene), the phosphoglycerate kinase 1 (PGK) promoter, the cytomegalovirus enhancer / promoter (CMV), the SV40 early promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter, the dihydrofolate reductase promoter, the P-actin promoter, and the EFl promoter. Musclespecific promoters include without limitation, upstream sequences derived from the desmin (Des) promoter, muscle creatine kinase (MCK) promoter or truncated muscle creatine kinase promoter (dMCK or tMCK), alpha-myosin heavy chain (alpha-MHC) promoter, myosin light chain 2 (MLC-2) promoter, cardiac troponin C (cTnC) promoter, the human skeletal actin (HSA) promoter or synthetic muscle-specific promoters such as SpC5-12 promoter, CK6 promoter, CK8 promoter. Various hepatocyte-specific promoters that can be used to express the transgene in hepatocytes and derived liver tissue or organ are well-known in the art and easily available, for example from the Liver Specific Gene Promoter Database compiled the Cold Spring Harbor Laboratory or other databases. The hepatocyte-specific promoter may be a promoter derived from any one of the following genes: Alpha- 1 -Antitrypsin (SERPINA1 or AAT), Albumin (ALB), Thyroxine-Binding Globulin (SERPINA7 or TBG), Transthyretin (TTR), Apolipoprotein Al (APOA1), Complement Factor B (CFB), Ketohexokinase (KHK), Hemopexin (HPX), Nicotinamide N-methyltransferase (NNMT), (liver) Carboxylesterase 1 (CES1), Protein C (PROC), Apolipoprotein C3 (APOC3), mannan-binding lectin serine protease 2 (MASP2), Hepcidin antimicrobial peptide (HAMP), Serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1), Fibrinogen Beta (FGB), Transferrin (TF), Insulin-Like Growth Factor II (IGF2) (promoter Pl), and Alpha-Fetoprotein (AFP). Alternatively, the promoter may be derived from a hepatitis virus, in particular human hepatitis virus such as HBV. A particular example of hepatitis virus promoter is HBV core promoter (CP HBV; Quarleri J., World J Gastroenterol., 2014, 20, 425-435.

[0060] (doi: 10.3748 / wjg.v20.i2.425). For example, the hepatocyte-specific promoter is selected from the group comprising: an alpha- 1 antitrypsin (SERPINA1 or AAT) promoter, a transthyretin (TTR) promoter, an albumin (ALB) promoter, a thyroxine-binding globulin (TBG) promoter and a Hepatitis B virus (HBV) core promoter. In some embodiments, the transgene is operably linked to a ubiquitous promoter as disclosed herein. In some embodiments, the transgene is operably linked to a muscle-specific promoter as disclosed herein. In some embodiments, the transgene is operably linked to a hepatocyte-specific promoter as disclosed herein.

[0061] AAV capsid

[0062] The AAV capsid may be of any AAV serotype. As used herein “AAV serotype” or “AAV capsid serotype” refers to an AAV capsid having distinct variable region (VR, also named hypervariable region or HVR) amino acid sequences compared to an AAV capsid of another serotype. Different AAV serotypes have amino acid variation in their VR sequences. The term AAV serotype encompasses any natural or artificial AAV capsid serotype including AAV capsid variants isolated from human or non-human species and AAV capsid variants engineered by various techniques known in the art such as for example rational design, directed evolution and in silico discovery. AAV serotype includes hybrid or chimeric AAV capsids. AAV serotype also includes peptide-modified AAV serotype derived from natural, variant, hybrid or chimeric AAV serotype. As used herein, the term AAV serotype refers to a functional AAV capsid which is able to form recombinant AAV viral particles which transduce a cell, tissue or organ, in particular a cell tissue or organ of interest (target cell, tissue or organ) and express a transgene in said cell, tissue or organ, in particular target cell tissue or organ.

[0063] In the context of AAV vectors, VR (Variable Region) sequences refer to specific regions within the viral capsid protein. These VR sequences play a critical role in determining the properties and behaviors of the AAV vector, including its productivity, tropism, transduction efficiency, interactions with target cells as well as its immunogenicity and neutralizing antibody escape. For example, using AAV serotype 8 (AAV8) capsid as reference sequence (VP1 of SEQ ID NO: 1 in WO 2022 / 003211), VR1 is from positions 146 to 153; VR2 is from positions 183-187; VR3 is from positions 263 to 267; VR4 is from positions 384 to 386; HVR5 is from positions 453 to 477; HVR6 is from positions 493 to 498; HVR7 is from positions 503 to 507; HVR8 is from positions 517 to 525; HVR9 is from positions 536 to 559; HVR10 is from positions 584 to 597; HVR11 is from positions 661 to 670; and HVR12 is from positions 708 to 722. VR3 to VR12 sequences are in the common C-terminal region corresponding to VP3 protein. A person skilled in the art can easily obtain the corresponding positions of the hypervariable regions in other AAV capsid serotypes after sequence alignment of any other AAV capsid sequence of any other serotype with AAV8 VP1 sequence using standard protein sequence alignment programs that are well-known in the art, such as for example BLAST, FASTA, CLUSTALW, MEGA and the like.

[0064] The AAV capsid serotype may be a variant, hybrid, and / or peptide-modified serotype. The AAV capsid serotype may be derived from natural AAV serotypes, variant AAV serotypes, hybrid AAV serotypes, peptide-modified AAV serotypes or combination thereof. Non limiting examples of natural AAV serotypes from which the AAV capsid may be derived include AAV1, AAV2 AAV3 (including types 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrhlO), AAV11, AAV12, AAVrh39, AAVrh43, AAVrh74, and AAVpol. Non limiting examples of variant AAV serotypes from which the AAV capsid may be derived include: AAV-DJ, AAV2i8, AAV-LK03, AAV-Anc80, AAV2G9, AAV.PHP (AAV-PHP.A, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S; AAV9BI; AAV9LD; AAVS1 and AAVS10 (El Andari et al., Science Advances, 2022, 8, 1-21; WO2019207132A1); AAV2 variants (WO2020 / 216861). Non limiting examples of hybrid AAV serotypes from which the AAV capsid may be derived include hybrid between AAV9 and AAVrh74, in particular AAV9.rh74 having the sequence SEQ ID NO: 3 as disclosed in WO2019 / 193119 and hybrid AAV capsids disclosed in WO 2022 / 003211. AAV8 capsid corresponds in particular to SEQ ID NO: 1 in WO 2022 / 003211. AAV serotype 9 (AAV9) capsid corresponds in particular to the amino acid sequence GenBank accession number AY530579.1 accessed on 24 June 2004. AAVrh74 corresponds in particular to SEQ ID NO: 2 in WO 2019 / 193119. Non limiting examples of peptide-modified AAV serotype from which the AAV capsid may be derived include AAV capsids comprising the insertion of a peptide comprising an RGD motif, which is known to bind several different cell-surface integrins, that have been reported to improve gene delivery in muscle following systemic administration. Peptides containing RGD motif are disclosed in Tabebordbar et al., Cell, 2021, 184, 4919-4938 and include: RGDLSTP (1C) (SEQ ID NO: 44), RGDLNQY (IB) (SEQ ID NO: 45), RGDLTTP (1 A) (SEQ ID NO: 46), RGDATEL (IF) (SEQ ID NO: 47), RGDQLYH (ID) (SEQ ID NO: 48), RGDVAAK (SEQ ID NO: 49), RGDTMSK (IE) (SEQ ID NO: 50), RGDMINT (1G) (SEQ ID NO: 51), RGDLNDS (SEQ ID NO: 52), RGDTMNY (SEQ ID NO: 53), 2A (GPGRGDQTTL) (SEQ ID NO: 54), 2B (AEGRGDQYTR) (SEQ ID NO: 55), 2C (ATGRGDLGQA) (SEQ ID NO: 56), 2D (AVARGDQGLI) (SEQ ID NO: 57), 2E (NISRGDQGYQ) (SEQ ID NO: 58), 2F (APARGDQGSQ) (SEQ ID NO: 59), 3A (RGDYVGL) (SEQ ID NO: 60), 3B (RGDYSGL) (SEQ ID NO: 61), 3C (RGDYSSV) (SEQ ID NO: 62), 3D (RGDYREL) (SEQ ID NO: 63), 3E (RGDHGVL) (SEQ ID NO: 64), 3F (RGDHASW) (SEQ ID NO: 65), 4A (SNSRGDYNSL) (SEQ ID NO: 66), 4B (STVRGDYTS) (SEQ ID NO: 67), 4C (QERRGDYTSM) (SEQ ID NO: 68), 4D (ASTRGDHGVL) (SEQ ID NO: 69), and 4E (ENRRGDFNNT) (SEQ ID NO: 70). The most efficient AAV capsids for muscle transduction in mice, non-human primates and / or human primary myotubes display a peptide comprising a RGDL motif : RGDLGLS (SEQ ID NO: 71) or Pl (AAVMYO or AAV9P1), RGDLTTP (SEQ ID NO: 72) (MyoAAV 1A), GPGRGDQTTL (SEQ ID NO: 73) (MyoAAV 2 A), SAQRGDYVGL (SEQ ID NO: 74) (MyoAAV 3A), SNSRGDYNSL (SEQ ID NO: 75) (MyoAAV 4 A), QERRGDYTSM (SEQ ID NO: 76) (MyoAAV 4C) and ENRRGDFNNT (SEQ ID NO: 77) (MyoAAV 4E) inserted into the variable region VIII (WO 2019 / 207132; Weinmann et al., Nature communications, 2020, 11, 5432; Tabebordbar et al., Cell, 2021, 184, 4919-4938). Other RGD peptides include integrin aVp6 binding peptides RGDLXXL / I (SEQ ID NO: 78) such as RGDLGRL (SEQ ID NO: 79) or RGDLATI (SEQ ID NO: 80) inserted into variable region IV (WO 2023 / 237748). Other peptides such as for example Al (MPLGAAG) (SEQ ID NO: 81) have been shown to improve gene delivery in muscle following systemic administration. Non-limiting examples of peptide modified AAV capsids include: AAVpol.Al (SEQ ID NO: 5 in WO 2021 / 219762); AAV9P1 or AAVMYO (SEQ ID NO: 12 in WO 2019 / 207132); AAVMYO2 and AAVMYO3 (SEQ ID NO: 30 and SEQ ID NO: 28 in WO 2019 / 207132) corresponding to Pl-modified AAVS1 and AAVS10 capsids respectively (El Andari et al. Science Advances, 2022, 8, 1-21). Pl -modified hybrid AAV9.rh74: AAV-MT (SEQ ID NO: 9 in WO 2019 / 193119) and AAV9.rh74-HB-Pl (SEQ ID NO: 6 in WO 2022 / 053630); LICA.01 (SEQ ID NO: 21 in WO 2023 / 237748). The AAV capsid protein may be VP1, VP2 or VP3 protein.

[0065] Method of preparation of barcoded AAV vectors The invention relates to a method of preparation of barcoded AAV vectors. In some embodiments, a method of preparation of a set of barcoded AAV vectors for assessing AAV vector transduction efficiency, in particular in vivo transduction efficiency, comprises the steps of: a) Providing n different AAV capsids, an AAV vector genome comprising a transgene expression cassette with a polyadenylation signal, and n different nucleic acid barcode sequences of at least 60 nucleotides according to the present disclosure; b) Inserting each nucleic acid barcode sequence, separately, into the AAV vector genome between the transgene stop codon and the polyadenylation signal; thereby generating n different barcoded AAV vector genomes that express a transgene transcript comprising one of the barcode sequences and a polyadenylation tail; and c) Encapsidating each barcoded AAV vector genome, separately with one of the AAV capsids to generate different barcoded AAV vector particles which are mixed to generate a set of n barcoded AAV vector particles.

[0066] The nucleic acid barcode sequence is inserted in the AAV vector genome between the transgene stop codon and the polyadenylation signal by standard molecular cloning techniques. For example, two different restriction sites may be inserted between the transgene stop codon and the polyadenylation signal; a DNA fragment comprising the nucleic acid barcode flanked in 5’ and 3’ with a copy of the restriction site may be generated by PCR and then digested with the restriction enzyme. The digested DNA fragment comprising the barcode sequence is then inserted at the unique restriction site located between the transgene stop codon and the polyadenylation signal to generate a barcoded AAV vector genome.

[0067] In some embodiments, n is from 2 to 10 (2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0068] The encapsidation step c) is performed using standard methods of AAV production that are well known in the art.

[0069] A number of production methods exist to generate AAV vectors (i.e. AAV particles) any of which may be used to prepare barcoded AAV vector particles according to the present disclosure. AAV vectors (i.e. AAV particles) are usually produced by standard co-transfection assays in appropriate cells for AAV production (see in particular Ayuso E. et al., Hum. Gene Ther. 2014, 25, 977-987). For example, human cell line HEK293 or derivatives such as HEK- 293T or HEK-293F cells are transfected with 3 plasmids: i) a transfer plasmid containing AAV2 ITRs flanking a transgene expression cassette (corresponding to AAV vector genome) ii) a helper plasmid containing adenoviral sequences (or helper function coming from other viruses) necessary for AAV production, and iii) a plasmid containing AAV Rep and Cap genes, defining the serotype of AAV. Alternatively, producer cells which stably express AAV Rep and Cap proteins may be transfected with an AAV transfer plasmid. Briefly, following transfection with above plasmid(s) in the presence of sufficient helper function to permit packaging of the rAAV vector genome into AAV capsid particle, the cells are incubated for a time sufficient to allow the production of AAV vector particles, the cells are then harvested, lysed, and AAV vector particles are purified by standard purification methods such as affinity chromatography and lodixanol or Cesium Chloride density gradient ultracentrifugation. Viral genomes are quantified by a TaqMan real-time PCR assay using primers and probes corresponding to the ITRs of the AAV vector genome (Rohr et al. J Virol Methods., 2002, 106, 81-8. doi: 10.1016 / s0166-0934(02)00138-6). rAAV titers are expressed as viral genome copy number (vg). Other methods can be used to produce AAV vectors (i.e. AAV particles) such as the insect cell / baculovirus system developed by Urabe et al. (J. Virol., 2006, 80, 1874- 1885) or the system using co-expression established in yeast (Barajas et al., PLoS One, 2017, 12, e0170010). Alternative system can also be used to produce AAV vectors (i.e. AAV particles), like production from hairy roots of a plant (WO 2021 / 123122).

[0070] The invention also relates to a barcoded AAV vector or a set of barcoded AAV vectors obtained by the method of preparation of barcoded AAV vectors according to the present disclosure.

[0071] The invention also relates to a kit for the preparation of a set of barcoded AAV vector, comprising at least n nucleic acid barcode sequences according to the present disclosure; preferably selected from the group consisting of SEQ ID NO: 1 to 7.

[0072] Method for assessing AAV vector transduction efficiency

[0073] The invention also relates to a method for assessing AAV vector transduction efficiency, in particular in vivo transduction efficiency, which is based on the detection of the nucleic acid barcode sequences according to the present disclosure. In some embodiments, the method for assessing AAV vector transduction efficiency, comprises: a) Contacting cells with a set of n barcoded AAV vector particles obtained by the method of preparation of barcoded AAV vectors according to the present disclosure; and b) Detecting the n barcode DNA and transcript sequences in the transduced cells obtained in step a) by quantitative polymerase chain reaction (PCR) and in situ hybridization using complementary oligonucleotide probes; and determining therefrom the AAV vector genome copy number and the transgene expression level and in situ detecting AAV vector transduced cells, for the different barcoded AAV vectors.

[0074] According to the method of the invention, the AAV vector genome copy number, the transgene expression level and the in situ detection of AAV vector in the transduced cells are determined for each one of the n different barcoded AAV vectors of the mixture of barcoded AAV vectors.

[0075] The contacting in a) may be performed in vitro or in vivo. Assessing in vitro transduction efficiencies may be performed on various cell systems that are well known in the art. Nonlimiting examples of cell systems include in vitro cell cultures (cell lines, primary cells, induced pluripotent cells, and others), ex vivo cell cultures, and various in vitro and ex vivo cell systems (organoids and others).

[0076] In some particular embodiments, a method for assessing AAV vector transduction efficiency in vivo, comprises: a) In vivo administration of a set n barcoded AAV vector particles obtained by the method of preparation of barcoded AAV vectors according to the present disclosure to at least one animal; and b) Detecting the n barcode DNA and transcript sequences in tissue sample(s) of the administered animal by quantitative polymerase chain reaction (PCR) and in situ hybridization using complementary oligonucleotide probes; and determining therefrom the AAV vector genome copy number and the transgene expression level and in situ detecting AAV vector transduced cells for the different barcoded AAV vectors.

[0077] In some embodiments, the AAV vector genome copy number is determined by quantitative real-time PCR (qPCR) or Droplet Digital PCR (ddPCR); the transgene transcript level is determined by real-time quantitative reverse transcription PCR (RT-qPCR) or reverse transcription Droplet Digital PCR (RT-ddPCR); and the in situ detection of AAV vector transduced cells is performed by in situ hybridization with signal amplification; preferably using SABER-FISH, USeqFISH, Zombie, RNAscope or BaseScope; preferably BaseScope.

[0078] RNAscope™ is disclosed in Wang et al., J. of Mol. Diagnostics, 2012, 14, 22-29. BaseScope™ is disclosed in Baker et al., Nat. Commun., 2017, 8, 1998 and Erben et al., Mol.. Neurobiol., 2018, 55, 6169-6181; Review in Erben, Curr. Protoc. Neurosci., 2019, 87, e63. SABER-FISH is disclosed in Wang et al., Molecular Therapy: Methods & Clinical Development, 2020, 19, 377. USeqFISH (Ultrasensitive sequential FISH) is disclosed in Jang et al., Nature Biotechnology, 2023, 41, 1206-1207 and 1272-1286. Zombie is disclosed in Askary et al., Nat. Biotechnol., 2020, 38, 66-75 and Mate Borsos et al., Molecular Therapy, 2021, Vol 29, No 4S1, Abstract 277).

[0079] The oligonucleotide probes for the quantitative polymerase chain reaction (PCR) and in situ hybridization may be the same or different.

[0080] In some embodiments, the oligonucleotide probes are selected from the group consisting of SEQ ID NO: 31 to 37. SEQ ID NO: 31 is specific for the barcode sequence of SEQ ID NO: 1; SEQ ID NO: 32 is specific for the barcode sequence of SEQ ID NO: 2; SEQ ID NO: 33 is specific for the barcode sequence of SEQ ID NO: 3; SEQ ID NO: 34 is specific for the barcode sequence of SEQ ID NO: 4; SEQ ID NO: 35 is specific for the barcode sequence of SEQ ID NO: 5; SEQ ID NO: 36 is specific for the barcode sequence of SEQ ID NO: 6; SEQ ID NO: 37 is specific for the barcode sequence of SEQ ID NO: 7. In some embodiments, these oligonucleotide probes are used at least for the quantitative polymerase chain reaction (PCR).

[0081] In some embodiments, the quantitative polymerase chain reaction uses oligonucleotide primers complementary to the barcode sequence; preferably selected from the group consisting of: SEQ ID NO: 11 to 24; more preferably from the following primer-pairs: SEQ ID NO: 11 and 12 (specific for the barcode sequence of SEQ ID NO: 1) ; SEQ ID NO: 13 and 14 (specific for the barcode sequence of SEQ ID NO: 2); SEQ ID NO: 15 and 16 (specific for the barcode sequence of SEQ ID NO: 3); SEQ ID NO: 17 and 18 (specific for the barcode sequence of SEQ ID NO: 4); SEQ ID NO: 19 and 20 (specific for the barcode sequence of SEQ ID NO: 5); SEQ ID NO: 21 and 22 (specific for the barcode sequence of SEQ ID NO: 6); and SEQ ID NO: 23 and 24 (specific for the barcode sequence of SEQ ID NO: 7).

[0082] In some embodiments, the method further uses antibodies specific for cell markers to determine the cell type(s) transduced by AAV vectors. Such markers are well-known in art. For example, anti-Cyp2el antibody may be used to label pericentral hepatocytes; anti -ALB antibody may be used to label periportal hepatocytes.

[0083] In some embodiments, the animal is a laboratory rodent such as mouse, or a non-human primate. Non-human primates include New World Monkeys such as marmosets and Old Word Monkeys such as Cynomolgus macaques, rhesus macaques and green monkeys. The animal is usually selected as seronegative for the AAV capsids tested.

[0084] In some embodiments, the analyzed tissues in step (b) include one or more of: liver; heart; skeletal muscles; brain; spinal cord; dorsal root ganglions; spleen; and kidney.

[0085] In some embodiments, the AAV vector transduced cell that’s is detected in situ is selected from the group consisting of: myocytes, myotubes, myoblasts, and / or satellite cells (for muscle tissue) ; neurons and glial cells, including oligodendrocytes, astrocytes, ependymal cells, and microglia (for CNS tissue); hepatocytes, liver endothelial cells, Kupffer cells, fatstoring cells (stellate cells or Ito cells), and pit cells (natural killer cells) (for liver tissue).

[0086] Another aspect of the invention relates to a kit for assessing AAV vector transduction efficiency, in particular in vivo transduction efficiency, comprising at least a set of oligonucleotide probes complementary to the barcode sequences according to the present disclosure; preferably selected from the group consisting of SEQ ID NO: 31 to 37. In some embodiments, the kit further comprises complementary oligonucleotide primers; preferably selected from the following pairs: SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO: 19 and 20; SEQ ID NO: 21 and 22; and SEQ ID NO: 23 and 24. The various embodiments of the present disclosure can be combined with each other and the present disclosure encompasses the various combinations of embodiments of the present disclosure.

[0087] The practice of the present invention will employ, unless otherwise indicated, conventional techniques, which are within the skill of art. Such techniques are explained fully in literature.

[0088] The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which:

[0089] FIGURE LEGENDS

[0090] Figure 1: Transduction assay

[0091] HeLa cells were transduced with QRcoded AAVmut5 vectors expressing hSeAP under CAG promoter, at MOI 32000. 48h post-transduction, medium was collected to measure hSeAP expression. No significant decrease of transgene expression is observed except for AAVmut5- QR#1 and QR#9. Data shown are means of hSeAP concentration in medium ± SD. Arrows indicate QRcoded leading to a decrease of transgene expression (i.e. QR#1 and QR#9).

[0092] Figure 2: VGCN and Transgene Expression in Liver

[0093] C57BL6 mice (n=3) were injected with QRcoded AAVmut5 vectors expressing hSeAP under CAG promoter at the dose of IxlO11vg total. On day 15, animals were sacrificed, and livers collected to assess VGCN and hSeAP RNA levels. Similar VGCN in liver is observed for 10 QRcoded AAVmut5. Similar transgene expression in liver is observed for AAVmut5-QR#2, -QR#3, -QR#4, -QR#5, -QR#6, QR#8 and -QR#10. A significant decrease of transgene expression is obtained for AAVmut5-QR#l, QR#7 and QR#9. Data shown are means of VGCN normalized mTitin gene ± SD and hSeAP RNA levels normalized RPLP0 ± SD. Red arrows indicate QRcoded AAVmut5 vectors leading to a decrease of transgene expression in liver (QR#1 QR#7 and QR#9).

[0094] Figure 3: Analysis of QR-code sequences a, Size of the selected barcodes and negative controls, b, Percentage of GC content in selected barcodes and negative controls, c, Gibbs free energy change (AG) in selected barcodes and negative controls expressed as kcal mol-1. Mean with standard deviation are presented for each group. Figure 4: VGCN and Transgene Expression in mouse injected with single or combined QRcoded AAVs a, Experimental protocol. C57B16 mice were injected with QRcoded AAV vectors expressing hSeAP under CAG promoter (AAV8, AAV9, AAVMT, AAV704, AAVmut2, AAVmut4 and AAVmut5) either in single vector (1x1011vg total) or in combined vectors (7x1011vg total total). On day 15, animals were sacrificed to collect tissues and blood samples, b, hSeAP expression in blood; c, e, Vector Genome Copy Number (VGCN) in Liver (c), and Triceps (e), from single and combined injections; d, f, Transgene Expression (hSeAP RNA levels) in Liver (d), and Triceps (f), from single and combined injections. Data shown are means of VGCN normalized mTitin gene ± SD and hSeAP RNA levels normalized RPLPO ± SD. g, h, in situ hybridization on liver from mouse injected with AAVmut5-QR#8 expressing hSeAP (g), and non-injected mouse (h). BaseScope probe used was specific for QR#8. Arrows show spots corresponding to hSEAP expression.

[0095] Figure 5: VGCN and Transgene Expression in NHP injected with combined QRcoded AAV vectors. Two male Cynomolgus macaques NHP1 and NHP2 were infused with a solution of QRcoded AAV vectors expressing hSeAP under CAG promoter (BOI-01 : AAV8, AAV9, AAVMT, AAV704, AAVmut2, AAVmut4 and AAVmut5; BOI-02: AAV8, AAVcbl3, AAVcb8, AAVC8-R10, AAVC8-R12, AAVC9-R5 and AAVmut5-1704) at the dose of 5xl012(vg)kg-l of each AAV vector). On day 15, animals were sacrificed to collect tissues and blood samples. a, c, e, g, Vector Genome Copy Number (VGCN) in Liver (a), Heart (c), Diaphragm (e) and Brain (g) from NHP1 and NHP2; b, d, f, h Transgene Expression (hSeAP RNA levels) in Liver (b), Heart (d), Diaphragm (f) and Brain (h) from NHP1 and NHP2; GCN and Transgene Expression of Liver were obtained from 2 wedge biopsies taken at different site of right / left, square and median lobes. VGCN and Transgene Expression of Brain were obtained from 2 biopsies of 4 Brain areas (frontal, occipital, trunk, cerebellum). Data shown are means of VGCN normalized eGlobin gene ± SEM and hSeAP RNA levels normalized RPLPO ± SEM. i, j, in situ hybridization on liver from NHP2. BaseScope probes used was specific for QR#8 corresponding to AAV9-hSeAP (i) and for QR#6 corresponding to AAVmut5-hSeAP (j). Arrows show spots corresponding to hSEAP expression.

[0096] EXAMPLES 1. Materials and Methods

[0097] 1.1 Care and use of animals

[0098] Mouse studies were performed according to the French and European legislation on animal care and experimentation and approved by the local institutional ethics committee (DAP 2016- 002-C, APAFIS#4654). All procedures using non-human primates (NHPs) were approved by the institutional animal care and use committee (APAFIS#33804-2021110815051790 and APAFIS#35575-2022022414273810) and were conducted at Nantes veterinary school (Oniris, Nantes, France).

[0099] 1.2 Mouse studies

[0100] Wild-type male C57BL / 6 mice male, aged 6 weeks, have received by tail vein route, either a single injection or combined injection of barcoded AAV-CAG-hSeAP vectors at the dose of 5xl012vg / kg of each vector (IxlO11vg / mouse). Blood was withdrawn with heparinized tubes via retro-orbital bleeding according to the schedule outlined in Figure 3a. At euthanasia, tissues and organs were collected for assessments of vector genome copy numbers (VGCN), hSeAP RNA levels and in situ hybridization.

[0101] 1.3 NHP studies

[0102] 4 Cynomolgus monkeys (Macaca fascicularis), 2-3 years old, were administered via slow intravenous infusion with combination of 7 barcoded AAV-CAG-hSeAP vectors at the dose of 5xl012vg / kg of each vector (i.e. total dose of 3.5xl013vg / kg); AAV8, AAV9, AAVMT, AAVmut2, AAVmut4, AAVmut5 or AAV8, AAVcbl3, AAVcb8, AAVC8-R10, AAVC8- R12, AAVC9-R5, AAVmut5-1704 for NHP1, NHP2 and NHP3, NHP4 respectively. Blood samples were collected throughout a protocol of 15 days. At sacrifice, several biopsies were withdrawn at different sites of organs and tissues; from the 4 liver lobes, right, left, caudate and papillary process; from 4 brain areas, frontal, occipital, trunk and cerebellum; from 3 heart areas, medio ventricular, apex, basal ; from diaphragm ; for assessments of vector genome copy numbers (VGCN), hSeAP RNA levels and in situ hybridization.

[0103] 1.4 Design of QRcoded expression cassettes

[0104] All QRcode sequences have been cloned in pGG21 expression cassette containing the CAG promoter, the chimeric intron, a kozak sequence, the hSEAP cDNA, and SV40 poly A signal. All cassettes were flanked by the ITRs of AAV serotype 2 for vector packaging. Moreover, a Multiple Cloning Site (MCS) was inserted after stop codon of hSeAP gene and before poly A signal, enabling the cloning of the QRcode sequences. By this way, 10 barcoded expressions cassettes were obtained, each containing a specific QRcode. The restriction profiles were checked and hSeAP expression were assessed by transfection to ensure that added barcode sequences did not impact the stability of hSeAP RNA transcripts. The QRcode sequences was synthesized by Genecust (Boynes, France).

[0105] 1.5 Production of QRcodedAAV vectors

[0106] All barcoded AAV vectors were produced using an adenovirus-free transient tri-transfection method in HEK293T cells growing in suspension and purified using a chromatographic method as described in Collaud F et al. Mol. Ther. Methods Clin. Dev. 2019. The following AAV capsids were tested: AAV8, AAV9, AAV-MT (WO 2019 / 193119); AAV704 (WO 2020 / 216861); AAVmut2, AAVmut4 and AAVmut5, AAVmut5-1704, AAVC8-R10 (AAV8-mut.HVR10), AAVC8-R12 (AAV8-mut.HVR12), and AAVC9-R5 (AAV9-R5- 704) disclosed in WO 2022 / 003211; AAVcbl3, AAVcb8. AAVcb8 and AAVcbl3 are AAV8 engineered capsids using rational shuffling method (WO 2022 / 003211 Al). AAVcb8 includes HVRs 1, 5 and 8 from the donor capsid AAV704; AAVcb8 includes HVRs 1, 5, 8 and 10 from the donor capsid AAV704.

[0107] AAV vectors were concentrated and reformulated in PBS1X, pluronic F68 0.001% using Amicon Ultra-15, 100k (Millipore). Titration of the barcoded AAV vectors was determined by TaqMan real-time quantitative PCR using primers specifics for ITRs (forward: 5Z-GGA ACC CCT AGT GAT GGA GTT-3’ (SEQ ID NO: 41); reverse: 5Z-CGG CCT CAG TGA GCG A-3Z(SEQ ID NO: 42); probe: 5Z-CAC TCC CTC TCT GCG CGC TCG-3 (SEQ ID NO: 43). Titer was expressed in vg / mL.

[0108] 1.6 Transduction assay

[0109] In 12-well plate, HeLa cells were seeded at the concentration of IxlO4cells / well in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% of fetal calf serum (FCS) and were incubated 45 minutes in 37°C, 5% CO2 incubator. Then, cells were transduced with QRcoded AAVmut5-hSeAP vectors at the multiplicity of infection (MOI) of 32000. After 48 hours, cell mediums were collected and hSeAP concentrations were measured. Quantification of hSeAP hSeAP was measured using the Phospha-Light™ Kit (Applied Biosystems) following the manufacturer’s instructions. Briefly, serum or medium samples were diluted in dilution buffer and incubated 30 minutes at 65°C. Then, samples were loaded on white 96-well microplate (OptiPlate-96, Perkin Elmer) and Assay Buffer was added in each well, following by the Reaction Buffer. After an incubation of 20 minutes, luminescence was measured using a microplate reader (ENSPIRE™, Perkin Elmer). The concentration of hSeAP was determined against a standard curve made from purified human placental alkaline phosphatase and results were expressed as pg / ml of serum or pg / ml of medium.

[0110] 1. 7 Vector Genome Copy Number (VGCN) determination

[0111] Whole-organ (mouse) or wedge biopsies (NHP) were homogenized using MN Beads tubes type D (Macherey Nagel) and Bead Mill 24 Homogenizer (Fisherbrand).

[0112] DNA was extracted from mouse tissues using NucleoMag Pathogen Kit (Macherey Nagel) and KingFisher Flex Device (Thermo Scientific). For NHP tissues, DNA was extracted using Gentra Puregen Blood Kit (Qiagen).

[0113] VGCN in tissues was determined by TaqMan real-time quantitative PCR using primers and probes specifics of barcode sequences (see Table 2a and 2b). Mouse titin and macaque globin were used as normalizing gene in mice and NHP studies, respectively. All primers and probes were purchased from Thermo Scientific.

[0114] Each sample was tested in duplicates and vector genome copy number per diploid genome was determined against a standard curve made of linearized plasmid. Results were means of VGCN per diploid genome.

[0115] In NHP study, VGCN were measured from 2 different biopsy samples withdrawn from different area of the organ or tissue. Means of VGCN + / - SEM were calculated from each area of organ tested. Results were represented on histogram, showing means VGCN + / - SEM in whole organ / tissue.

[0116] 1.8 Transgene RNA levels determination

[0117] Whole-organ (mouse) or wedge biopsies (NHP) were homogenized using MN Beads tubes type D (Macherey Nagel) and Bead Mill 24 Homogenizer (Fisherbrand).

[0118] Tissues were lysed in NucleoZOL (Macherey Nagel) and total RNA were extracted using NucleoMag Kit (Macherey Nagel) and IDEAL™ 32 extraction robot (Innovative Diagnostics). Reverse transcription was performed on RNA extracts previously treated with Turbo DNase (life Technologies), using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). hSeAP RNA level was determined by TaqMan real-time quantitative PCR using primers and probes specifics of barcode sequences. RPLPO was used as normalizing gene in mice and NHP studies. Each sample was tested in duplicates and hSeAP and RPLPO RNA levels were determined against standard curves made of linearized plasmids.

[0119] For NHP study, transgene RNA level was measured from 2 different biopsies withdrawn from different area of the organ or tissue. Mean of hSeAP RNA levels + / - SEM was calculated from each area of organ tested. Results were represented on histogram, showing means hSeAP RNA levels + / - SEM in whole organ / tissue.

[0120] 1.9 In situ hybridization

[0121] In situ hybridization (ISH) was used to detect the presence of transgene RNA in AAV-injected mouse and NHP liver tissues. ISH was performed using the BaseScope Duplex detection reagent kit (Cat. No. 323810) and the BaseScope Duplex probes for channel 1 and 2. Cell markers were labelled in channel 1 (Cl) and AAV vector transgene (hSEAP) in channel 2 (C2). Cyp2el (Ref. 1103361-C1 for mouse liver; ref. 1103371-C1 for NHP liver) and ALB (Ref. 1098001 -Cl for mouse liver; ref. 1104021 -Cl for NHP liver) probes were used to stain pericentral and periportal hepatocytes respectively. To detect hSEAP RNA expression, custom made C2 probes specific for the 7 QR-code sequences were designed by Bio-Techne. BaseScope experiment was performed on formalin-fixed paraffin-embedded (FFPE) tissues following the manufacturing protocol. Each QR-code specific C2 probe was analyzed in combination with cell specific Cl probes.

[0122] 1.10 Analysis of the Gibbs free energy change

[0123] Gibbs free energy change (AG) is the energy required to break the secondary structure, and larger negative values indicate a higher propensity of the DNA to form stable intramolecular structure. To calculate the AG of the barcode, we used the DNA Secondary Structure online Prediction Tool by Vector Builder.

[0124] 2. Results A barcoded method was developed to assess the in vivo efficiency of new engineered capsids in mouse and non-human primate (NHP). First, 10 potential barcode DNA sequences of around 80bp having no match with mouse and NHP genomic DNA were selected and named QRcode (Table 1) Table 1: AAV barcodes

[0125] Primers and probes specific for the QRcodes were designed (Table 2a and 2b).

[0126] Table 2a: Primers specific for the AAV barcodes

[0127] Table 2b: Probes specific for the AAV barcodes The barcode DNA sequences were cloned into CMV early enhancer / chicken P actin (CAG) promoter / human secreted embryonic alkaline phosphatase (hSEAP) transgene (CAG-hSeAP) expression cassette between transgene stop codon and polyA to produce AAV vectors. In vitro and in vivo experiments showed that 7 DNA sequences (QR#2, QR#3, QR#4, QR#5, QR#6, QR#8 and QR#10) had no impact on transgene expression and allowed the production of 7 AAV variants, each one carrying a specific QRcode (Figures 1 and 2).

[0128] The seven selected barcode sequences were analyzed and compared to that of negative controls (QR#7, QR#9, QR#1); Table 3 and Figure 3. They have a size of 60 nt to 100 nt and most of them (6 / 7) have an intramolecular structure stability, AG superior or equal to -27 kcal mol’1; 5 / 7 have a AG superior or equal to -15 kcal mol’1. More than half of the best performing barcode sequences have a GC content of less than 50 %.

[0129] Table 3: Barcode characteristics

[0130] C57bl6 mice were injected with single or combined vectors, and AAVs biodistribution and transgene expression on different tissues was assessed by qPCR using QRcode specific probe sets (Figure 4a). As expected, different concentration of hSeAP in blood (Figure 4b) as well as different biodistribution (VGCN) and hSeAP RNA profiles were obtained according to tissue specificity of each barcoded AAV variants but no significant difference between single and combined vectors injections were observed (Figures 4c, 4d, 4e, 4f). Thanks to the large size of QRcode DNA, it was possible to perform in situ Hybridization with BaseScope technique on liver tissue which allows to analyze transgene expression of the different AAVs according to liver zonation (Figures 4g and 4h). Finally, the QRcode method was validated in NHP co-injected with 7 QRcoded AAV capsids (Figures 5a, 5b). The analysis of biodistribution and transgene expression in different tissues confirmed the reliability and the robustness of the method for the characterization of new AAV vectors (Figures 5c, 5d, 5e, 5f). In contrast with classical barcoded library approaches, the QRcode method allows to assess the vector biodistribution of selected AAV variants at medium-throughput by classical qPCR or droplet digital PCR and to characterize vectors localization in the tissue by in situ hybridization (Figures 5i, 5j)

Claims

CLAIMS1. A method of preparation of a set of barcoded AAV vectors for assessing AAV vector transduction efficiency, comprising the steps of: a) Providing n different AAV capsids, an AAV vector genome comprising a transgene expression cassette with a polyadenylation signal, and n different nucleic acid barcode sequences of at least 60 nucleotides, wherein each barcode sequence is detectable by both in situ hybridization and quantitative polymerase chain reaction using complementary oligonucleotide probe(s), wherein the nucleic acid barcode sequence is a DNA barcode sequence selected from the group consisting of SEQ ID NO: 1 to 7; b) Inserting each nucleic acid barcode sequence, separately, in the AAV vector genome between the transgene stop codon and the polyadenylation signal; thereby generating n different barcoded AAV vector genomes that express a transgene transcript comprising one of the barcode sequences and a polyadenylation tail; and c) Encapsidating each barcoded AAV vector genome, separately with one of the AAV capsids to generate different barcoded AAV vector particles which are mixed to generate a set of n barcoded AAV vector particles.

2. The method of claim 1, wherein the AAV capsids are from variant AAV serotypes, hybrid AAV serotypes, peptide-modified AAV serotypes, or combination thereof.

3. The method of claim 1 or 2, wherein n is from 2 to 10.

4. A barcoded AAV vector or a set of barcoded AAV vectors obtained by the method according to any one of claims 1 to 3 comprising a DNA barcode sequence selected from the group consisting of SEQ ID NO: 1 to 7.

5. A method for assessing AAV vector transduction efficiency, comprising: a) Contacting cells with a set of n barcoded AAV vector particles obtained by the method of preparation of barcoded AAV vectors according to any one of claims 1 to 3; andb) Detecting the n barcode DNA and transcript sequences in the transduced cells obtained in step a) by quantitative polymerase chain reaction (PCR) and in situ hybridization using complementary oligonucleotide probes; and determining therefrom the AAV vector genome copy number and the transgene expression level and in situ detecting AAV vector transduced cells, for each one of the n barcoded AAV vectors.

6. The method of claim 5 for assessing AAV vector transduction efficiency in vivo, comprising: a) In vivo administration of a set n barcoded AAV vector particles obtained by the method according to any one of claims 1 to 3 to at least one animal; and b) Detecting the n barcode DNA and transcript sequences in tissue sample(s) of the administered animal by quantitative polymerase chain reaction (PCR) and in situ hybridization using complementary oligonucleotide probes; and determining therefrom the AAV vector genome copy number and the transgene expression level and in situ detecting AAV vector transduced cell for each one of the n barcoded AAV vectors.

7. The method according to claim 5 or 6, wherein the oligonucleotide probes are selected from the group consisting of SEQ ID NO: 31 to 37.

8. The method according to any one of claims 5 to 7, wherein the AAV vector genome copy number are determined by quantitative real-time PCR (qPCR) or Droplet Digital PCR (ddPCR); the transgene transcript level are determined by real-time quantitative reverse transcription PCR (RT-qPCR) or reverse transcription Droplet Digital PCR (RT-ddPCR); and the in situ detection of AAV vector transduced cells is performed by in situ hybridization with signal amplification; preferably using SABER-FISH, Zombie, USeqFISH, RNAscope or BaseScope; preferably BaseScope.

9. The method according to any one of claims 5 to 8, which uses complementary oligonucleotide primers; preferably selected from the following pairs: SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO: 19 and 20; SEQ ID NO: 21 and 22; and SEQ ID NO: 23 and 24.

10. The method according to any one of claims 6 to 9, wherein the animal is a laboratory rodent such as mouse, or a non-human primate.

11. The method according to any one of claims 6 to 10, wherein the analyzed tissues in step (b) include one or more of: liver, heart, skeletal muscles, brain, spinal cord, dorsal root ganglions, spleen, and kidney.

12. Akit for the preparation of a set of barcoded AAV vectors, comprising at least n nucleic acid barcode sequences selected from the group consisting of SEQ ID NO: 1 to 7.

13. A kit for assessing AAV vector transduction efficiency comprising at least a set of oligonucleotide probe(s) complementary to the barcode sequences selected from the group consisting of SEQ ID NO: 1 to 7; preferably selected from the group consisting of SEQ ID NO: 31 to 37; preferably wherein the kit further comprises complementary oligonucleotide primers; preferably selected from the following pairs: SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO: 19 and 20; SEQ ID NO: 21 and 22; and SEQ ID NO: 23 and 24.

14. A nucleic acid barcode selected from the group consisting of SEQ ID NO: 1 to 7.

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