Viral vector for transduction of adipocytes

Abstract

Viral vectors based on adeno-associated virus (AAV), preferably AAV serotype 1 (AAV1), are provided for use in transduction of adipocytes. The viral vectors of the invention have the advantage of being adapted in tropism for adipocytes, wherein the viral vectors especially in use for intraperitoneal administration are optimized for adipocytes residing in SCAT (subcutaneous adipose tissue) or in VAT (visceral adipose tissue), or the viral vectors in use for oral administration are optimized for adipocytes residing in SCAT or in VAT.

Description

[0001] Viral vector for transduction of adipocytes

[0002] The present invention relates to viral vectors based on adeno-associated virus (AAV), preferably AAV serotype 1 (AAV1), for use in transduction of adipocytes, especially for use in in vivo or in ex vivo transduction of adipocytes, especially for genetic manipulation of adipocytes. The viral vectors of the invention have the advantage of being adapted in tropism for adipocytes, wherein the viral vectors especially in use for intraperitoneal administration are optimized for adipocytes residing in SCAT (subcutaneous adipose tissue, also termed subcutaneous white adipose), or the viral vectors in use for intraperitoneal administration are optimized for adipocytes residing in VAT (visceral adipose tissue, also termed visceral white adipose), or the viral vectors in use for oral administration are optimized for adipocytes residing in SCAT, or the viral vectors in use for oral administration are optimized for adipocytes residing in VAT.

[0003] Accordingly, the viral vectors of the invention are engineered for targeting and transducing adipocytes of SCAT or adipocytes of VAT, in each case either by intraperitoneal administration or by oral administration. The viral vectors of the invention have the advantage of being optimized for transducing adipocytes with specificity for SCAT or VAT, which specificity is also dependent on and can be controlled by the route of administration, and therefore are suitable for use in medical treatment by expressing a nucleic acid construct in adipocytes of SCAT or VAT. Adipocytes and adipose tissue as a target of the viral vectors of the invention have the advantage that no tumours are known that originate from adipose tissue, no fibrogenesis is known to originate from adipose tissue, generally, adipose tissue can be removed without an organ loosing its function, and in comparison to e.g. liver immune responses against adipose tissue are generally low due to low presence of antigen presenting cells in adipose tissue, and genetic manipulation of adipose tissue is expected to be long-lived, e.g. stable, as adipocytes are nonproliferating and long-lived.

[0004] State of the art

[0005] Bates et al., Molecular Therapy: Methods & Clinical Development Vol. 19, 236-249 (2020) review AAV-based vectors for targeting adipose tissue, quoting low transduction efficiency for AAV1 even when directly injected into adipose tissue.

[0006] Hacker et al., The Journal of Gene Medicine 2005; 7: 1429-1438 describes a self- complementary AAV vector genome configuration.

[0007] Object of the invention

[0008] It is an object of the invention to provide viral vectors having tropism for adipocytes, suitable for transducing adipocytes, wherein preferably the viral vectors have specificity for SCAT or VAT. More preferred, the viral vectors shall have optimized transduction abilities for SCAT or VAT, depending on the route of administration, e.g. depending on oral or intraperitoneal administration.

[0009] Description of the invention

[0010] The invention achieves the object by the features of the claims and especially provides a viral vector, preferably an AAV vector, more preferably an AAV1 vector, which in the VP1 reading frame of the capsid ORF (capsid protein) C-terminally to amino acid No. 590, which is aspartate (D), contains an insert comprising one of SEQ ID NO: 1 to SEQ ID NO: 80, wherein preferably the insert is flanked by 2 to 3 amino acid residues as a linker, preferably alanine residues, e.g. 3 alanine residues between amino acid No. 590 and the insert of one of SEQ ID NO: 1 to SEQ ID NO: 80, and C-terminally to the insert contains 2 alanine residues as a linker, with the remainder of the VP1 reading frame arranged C-terminally to these alanine residues. The insert, preferably flanked both N-terminally and C-terminally by a linker of 2 to 3 amino acids as a linker, e.g. 2 to 3 alanine residues, is arranged between amino acid No. 590 and amino acid No. 591 of the wild-type sequence of VP1 (virion protein 1, SEQ ID NO: 81) reading frame of the capsid ORF, e.g. of AAV1. The capsid of the invention, which contains one of the inserts, which insert is flanked by an N-terminal linker of 3 alanine residues (amino acids 591..593) and a C-terminal linker of 2 alanine residues (amino acids 601..602) is represented by SEQ ID NO: 82, wherein the insert is represented by a section of seven wildcard amino acids (No. 594..600). Accordingly herein, each of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 80 can replace the section of the seven wildcard amino acids of SEQ ID NO: 82. The viral vector of the invention is suitable for use in medical treatment of adipocytes, especially for transduction adipocytes, e.g. transducing adipocytes with a nucleic acid construct, which e.g. contains a nucleic acid section for recombination with chromosomal DNA of an adipocyte, a nucleic acid encoding a protein, and / or a nucleic acid encoding an RNA, e.g. encoding an inhibitory RNA, an mRNA, a long noncoding RNA (IncRNA) or a microRNA (miRNA), or other small RNAs for use in transcriptional gain-of- function or loss-of-function methods, e.g. single guide RNA (sgRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) or other small RNAs using in transcriptional gain- or loss-of-function approaches (single guide RNA, small interfering RNA, short hairpin RNA), or for use of the AAV vector for the transfer of genome editing compounds and / or nucleic acid sequences as templates for use of the homology-directed repair (HDR).

[0011] Generally, the process for producing AAV-based viral vector particles according to the invention can be by delivery, e.g. by plasmid transfection with or without helper virus coinfection, of all required components for AAV vector production, e.g. from a vector genome containing the transgene expression cassette flanked by ITRs or a nucleic acid sequence for HDR, AAV rep and AAV cap genes as well as other viral helper genes necessary for AAV particle production, e.g. from adenovirus. The process can be performed in a cultivated eukaryotic host cell, followed by cell lysis and removal of cellular components and plasmid DNA, e.g. by enzymatic digestion, filtration and / or centrifugation, and further purification, e.g. by gradient density centrifugation and / or chromatography of AAV viral vector particles. In a specific embodiment, empty viral particles comprising the protein of the VP1 reading frame of the capsid ORF (capsid protein) with the insert according to the invention are provided, which do not contain a nucleic acid molecule. These empty viral particles can e.g. be associated with a functional molecule for delivery of the functional molecule to adipocyte tissue, preferably specifically to SCAT or VAT. The functional molecule can e.g. be a therapeutic agent or an indicator compound, e.g. a dye or a pharmaceutically acceptable diagnostic contrast agent, or a combination of at least two of these. Empty viral particles can be produced e.g. in HEK293 cells. The process comprises the steps of transfecting the respective helper plasmid (containing the cap gene, if applicable with an inserted amino acid section, and the rep gene of wild-type AAV1) and an adenoviral helper plasmid (containing adenoviral helper functions required for AAV vector production) in HEK293 cells for AAV empty capsid production (no vector genome plasmid containing ITRs flanking the expression cassette is used in this specific case), with subsequent purification of empty capsid particles by iodixanol gradient centrifugation.

[0012] Preferably, the AAV vector, preferably the AAV 1 vector, for use in intraperitoneal administration for transduction of adipocytes residing in SCAT contains an insert of one of SEQ ID NO: 1 to SEQ ID NO: 20, preferably SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; the AAV vector, preferably the AAV1 vector, for use in intraperitoneal administration for transduction of adipocytes residing in VAT contains an insert of one of SEQ ID NO: 21 to SEQ ID NO: 40, preferably SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; the AAV vector, preferably the AAV1 vector, for use in oral administration for transduction of adipocytes residing in SCAT contains an insert of one of SEQ ID NO: 41 to SEQ ID NO: 60, preferably SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43; and the AAV vector, preferably the AAV1 vector, for use in oral administration for transduction of adipocytes residing in VAT contains an insert of one of SEQ ID NO: 61 to SEQ ID NO: 80, preferably SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63.

[0013] Most preferred, the AAV vector, preferably the AAV1 vector, contains one of SEQ ID NO: 61 (also termed VP48), SEQ ID NO: 62 (also termed VP51), SEQ ID NO: 41 (also termed VP50), SEQ ID NO: 42 (also termed VP49), and SEQ ID NO: 63 (also termed VP47), each for use in oral administration. Accordingly, the invention also provides a method for transducing adipocytes residing in adipose tissue by the AAV vector of the invention, e.g. adipocytes residing in SCAT and / or in VAT, preferably a method for medical treatment. Therein, the treatment by transducing adipocytes residing in adipose tissue may comprise the transduction with a nucleic acid construct contained the AAV vector, e.g. a nucleic acid section for recombination with chromosomal DNA of an adipocyte, a nucleic acid encoding a protein, and / or a nucleic acid encoding an RNA, e.g. encoding an inhibitory RNA, an mRNA, a long noncoding RNA (IncRNA) or a microRNA (miRNA), or other small RNAs for use in transcriptional gain-of- function or loss-of-function methods, e.g. single guide RNA (sgRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) or other small RNAs using in transcriptional gain-of- function or loss-of-function approaches (single guide RNA, small interfering RNA, short hairpin RNA), or for use of the AAV vector for the transfer of genome editing compounds and / or nucleic acid sequences as templates for use of the homology-directed repair (HDR).

[0014] Accordingly, the invention also provides an in vitro method for transducing adipocytes which originate from adipose tissue, e.g. SCAT and / or VAT, for introducing a nucleic acid construct into the adipocytes, optionally with subsequent introduction of transduced adipocytes into a human, e.g. for medical treatment of a human by administration, e.g. transplantation, of adipocytes genetically manipulated by transduction by an AAV vector of the invention, e.g. for expression of a nucleic acid construct or for expression of a protein encoded by the nucleic acid construct transduced by the AAV vector.

[0015] Generally, the vector of the invention can contain a nucleic acid construct which encodes an RNA or a protein for extracellular expression, e.g. for transduced adipocytes secreting the RNA or protein encoded by a nucleic acid construct transduced by the viral vector.

[0016] The invention is now described in greater detail by way of examples and with reference to the figures, which show in

[0017] - Fig. 1 A relative transduction levels of murine adipocytes by viral vectors of the invention, indicated by expression levels of GFP,

[0018] - Fig. 1B-G micrographs of cells transduced with viral vectors expressing GFP,

[0019] - Fig. 2A relative transduction levels of human adipocytes by viral vectors of the invention, indicated by expression levels of GFP,

[0020] - Fig. 2B-G micrographs of cells transduced with viral vectors expressing GFP, - Fig. 3 results for transduction strength of comparative AAV8,

[0021] - Fig. 4 results for transduction specificity of comparative AAV8,

[0022] - Fig. 5 results for transduction strength of AAV 1 of the invention relative to AAV8,

[0023] - Fig. 6 results for transduction specificity of AAV1 of the invention,

[0024] - Fig. 7 results for transduction strength of AAV1 of the invention relative to AAV8,

[0025] - Fig. 8 results for transduction specificity of AAV1 of the invention,

[0026] Fig. 9 results for tropism for liver of AAV1 of the invention and for comparative AAV8, and in

[0027] - Fig. 10 results for quality control of AAV1 of the invention and for comparative AAV8.

[0028] Example 1 : Selection of AAV1 viral particles with tropism for adipocytes from an AAV1 peptide display library

[0029] A library of AAV1 viral particles containing more than 106different inserts of 7 random amino acids arranged in the VP1 capsid protein was constructed. In short, the coding sequence for the wild-type capsid protein of SEQ ID NO: 81, containing a linker of three alanine residues - seven random amino acids - a linker of two alanine residues between amino acids No. 590 and 591 of the wild-type capsid proteins, corresponding to amino acids 590 and 603 of SEQ ID NO: 82, with the seven random amino acids comprising more than 106different inserts was constructed. This library was cloned as plasmid library by inserting oligonucleotides of random sequence flanked by sequences coding for a linker of three and two alanine residues, respectively, into the cap gene at a position which is between the amino acids corresponding to the amino acid positions 590 and 591 of the wild-type capsid. The plasmid library was used to produce the viral AAV peptide display library in HEK293 cells. Following an initial production step, the resultant capsid engineered viral particles were subjected to a phenotype-genotype coupling step to enable identification of the capsid modification through sequencing of the cap gene delivered by the AAV particles to the target cells.

[0030] The viral particles were produced in HEK producer cells. In short, the variants were produced as viral vectors in HEK293 cells using an adenoviral helper plasmid (e.g. pXX6), a vector plasmid encoding for eGFP in a self-complementary vector genome configuration and the respective helper plasmid encoding for AAV2 rep and the modified AAV1 cap gene. For each variant a unique AAV helper plasmid was used. Following vector production, cells here harvested and lysed. Viral vector particles were purified by density gradient centrifugation followed by buffer exchange.

[0031] Genomic particle titer was determined by qPCR. Subsequently, the library was subjected towards a phenotype-genotype coupling step. For this, HEK293 were seeded on top of viral particles bound to a cell culture plate by anti-AAVl antibody. This step enables uptake of AAV particles independent of a specific targeting receptor. Due to performing this coupling step at a parti cle-per-cell ratio of approximately 1000, only limited number of viral genomes are entering the cell nucleus ensuring formation of uniform capsids and packaging of the cognate viral genome. Following production, the library is again subjected to a density gradient centrifugation and determination of genomic particle titers by qPCR. The titer of the library was 7xl09per microliter.

[0032] Viral particles containing the different inserts were administered to C57BL / 6N mice, once by intraperitoneal injection of 3.5 x IO10viral particles per mouse suspended in 1 x PBS (intraperitoneal administration), once by oral administration of 3.5 x IO10viral particles diluted in 20% sucrose solution.

[0033] Ten days subsequent to the intraperitoneal or oral administration, mice were sacrificed and subcutaneous white adipose tissue (SCAT) and visceral white adipose tissue (VAT) were isolated. The isolated SCAT and VAT were used to isolate mature (buoyant) floating adipocytes. From these purified adipocytes, total genomic DNA was isolated, followed by next generation sequencing (NGS) in order to identify the sequences encoding the insert introduced into each of the capsid subunits at VP1 C-terminally to amino acid 590, which insert mediated infection of adipocytes.

[0034] In total, in SCAT for intraperitoneal administration, 38715 sequences were analysed, identifying a total of 4173 different insert sequences, in VAT for intraperitoneal administration, 53039 sequences were analysed, identifying a total of 9173 different insert sequences, in SCAT for oral administration, 15189 sequences were analysed, identifying a total of 324 different insert sequences, in VAT for oral administration, 27429 sequences were analysed, identifying a total of 470 different insert sequences. It was found that viral particles with VP1 capsid proteins containing specific inserts were enriched in either SCAT or VAT in dependence on the route of administration. VP1 capsid proteins containing the insert sequences were ranked according to their proportion, indicating their specificity for SCAT or VAT :

[0035] This result shows that from the high number of VP1 capsid proteins having different inserts that were administered, after the in vivo selection, the capsid proteins containing one of these inserts show tropism for adipose tissue. Interestingly, the route of administration had a strong influence on the tropism for VAT or SCAT. Accordingly, for in vivo use, an AAV1 vector particle having a VP1 containing an insert of one of SEQ ID NO: 1 to SEQ ID NO: 20 is preferred for intraperitoneal administration for transducing SCAT, an AAV1 vector particle having a VP1 containing an insert of one of SEQ ID NO: 21 to SEQ ID NO: 40 is preferred for intraperitoneal administration for transducing VAT, an AAV1 vector particle having a VP1 containing an insert of one of SEQ ID NO: 41 to SEQ ID NO: 60 is preferred for oral administration for transducing SCAT, and an AAV1 vector particle having a VP1 containing an insert of one of SEQ ID NO: 61 to SEQ ID NO: 80 is preferred for oral administration for transducing VAT, wherein preferably the insert is contained in a VP1 according to SEQ ID NO: 82, therein replacing the wildcard amino acid residues.

[0036] Example 2: Transduction of adipocytes by AAV1 viral vector particles and capsid modified AAV1 derived viral vector particles

[0037] As representatives of the AAV vector particles, which preferably are AAV1 viral vector particles, of the invention, viral vector particles having the VP1 protein according to SEQ ID NO: 82 with an insert of one of SEQ ID NO: 63 (VP47), SEQ ID NO: 61 (VP48), SEQ ID NO: 42 (VP49), SEQ ID NO: 41 (VP50), and SEQ ID NO: 62 (VP51) replacing the seven wild-card amino acids, were used to transduce in vitro cultivated adipocytes. As a representative and indicator for successful transduction, the viral particles contained an expression cassette for enhanced GFP (eGFP) in a self-complementary AAV vector genome configuration (as described by Hacker et al., The Journal of Gene Medicine 2005; 7: 1429- 1438). In short, murine or human adipocytes were cultivated in standard DMEM medium to confluence, after which adipogenesis was induced by adding IpM rosiglitazone, 850nM insulin, IpM dexamethasone, and 250pM IB MX for a duration of eleven days. After successful adipocyte differentiation, mature adipocytes were treated with 50,000 (designation are indicated in Fig. 1) viral particles per cell for transduction to the cell culture and cells were harvested for immunofluorescence measurements and / or quantitative RT-PCR after 48h.

[0038] Fig. 1 A shows data for expression of eGFP measured 48 h after transduction in murine adipocytes by qPCR using transgene specific primers and following normalization to levels of b-actin mRNA. Murine adipocytes were incubated with indicated vectors at a particle-per-cell ratio of 50 000. Cells were lysed and mRNA was isolated 48 hrs post vector administration. qPCR analysis was performed on cDNA of samples and shown are results normalized to housekeeping gene b-actin. Specifically, these results reveal that the preferred insert of VP51 and insert VP48, and to a lower extent the insert VP47 and VP49 efficiently transduced murine adipocytes for expression of a transgene encoded by a nucleic acid construct contained in an AAV1 particle, whereas the wild-type capsid (CTRL; AAV1) in comparison shows only background transduction activity. Figures 1B-E show micrographs of the cells expressing GFP after transduction with the viral vector particles having the VP1 protein according to SEQ ID NO: 82 with an insert of one of SEQ ID NO: 63 (VP47) in Fig. IB, SEQ ID NO: 61 (VP48) in Fig 1C, SEQ ID NO: 42 (VP49) in Fig. ID, SEQ ID NO: 41 (VP50) in Fig. IE, and SEQ ID NO: 62 (VP51) in Fig. IF, for comparison wild-type AAV1 in Fig. 1G.

[0039] Fig. 2A shows data for expression of eGFP measured in human adipocytes by qPCR using transgene specific primers and following normalization to levels of b-actin mRNA. Murine adipocytes were incubated with indicated vectors at a particle-per-cell ratio of 50 000. These results reveal that the preferred insert of VP48, and to a lower extent the insert VP47, VP49, VP50 and VP51 efficiently transduce human adipocytes for expression of a transgene encoded by a nucleic acid construct contained in an AAV1 particle, whereas the wild-type capsid (CTRL; AAV1) in comparison shows only background transfection activity. The micrographs of Fig. 2A-G show adipocytes transduced with the viral vector particles having the VP1 protein according to SEQ ID NO: 82 with an insert of one of SEQ ID NO: 63 (VP47) in Fig. 2B, SEQ ID NO: 61 (VP48) in Fig 2C, SEQ ID NO: 42 (VP49) in Fig. 2D, SEQ ID NO: 41 (VP50) in Fig. 2E, and SEQ ID NO: 62 (VP51) in Fig. 2F, for comparison wild-type AAV1 in Fig. 2G.

[0040] Example 3: In vivo transduction of adipose tissue by AAV vectors

[0041] As examples for in vivo transduction showing expression of a reporter protein as a model nucleic acid construct encoded by viral vectors of the invention, AAV1 containing in its VP1 the insert of SEQ ID NO: 61 (VP48) or the insert of SEQ ID NO: 62 (VP51), or as a comparison AAV8 were administered to 4 male C57BL / 6 mice each, by intravenous administration (intravenous RoA), or by intraperitoneal administration (intraperitoneal RoA), or by subcutaneous administration (subcutaneous RoA), using a total of 2 x 1011vg (viral genomes) in saline per animal. Each viral vector contained and expression cassette encoding the green fluorescent protein ZsGreenl under the control of the constitutive strong CMV promoter (CMV) as a model nucleic acid construct for expression in transduced cells.

[0042] Mice were sacrificed 29 days subsequent to administration of the viral vector and dissected for analysis of expression of ZsGreenl in different tissues. ZsGreenl expression was measured by digital quantitative PCR (qPCR) of RNA encoding ZsGreenl. In the Figures, results for ZsGreenl expression is depicted for liver anterior lobe (Anterior lobe, Ant lobe), for liver posterior lobe (Posterior lobe), for liver median lobe (Median lobe), for liver left lobe (Left lobe), for subcutaneous white adipose tissue (scWAT or SCAT), for brown adipose tissue (BAT), for gonadal visceral white adipose tissue (gWAT or VAT) and for inguinal white adipose tissue (ingWAT).

[0043] Fig. 3 shows results for transduction strength of AAV8 containing the expression cassette for ZsGreenl as a comparative viral vector (AAV8-CMV-ZsGreenl), the transduction strength being calculated relative to expression of ZsGreenl in liver anterior lobe after administration of the comparative viral vector (AAV8-CMV-ZsGreenl) by i.v. administration (AAV8 I.V. Liver (Ant lobe). As shown, transduction of the expression cassette differs in different tissues and differs by route of administration.

[0044] Fig. 4 results for transduction specificity of comparative AAV8-CMV-ZsGreenl, relative to expression in liver anterior lobe (Liver Ant lobe), determined for each individual route of administration. As shown, transduction specificity for different tissues differs by route of administration.

[0045] Fig. 5 shows results for transduction strength of AAV1 with the insert VP48 of the invention and containing the same expression cassette for ZsGreenl (AAVl-VP48-CMV-ZsGreenl). Data are shown relative to expression of ZsGreenl as transduced by comparative AAV8- CMV-ZsGreenl. These results show that this AAV with insert VP48 after intravenous administration only results in very low expression of the reporter, after intraperitoneal administration results in high expression specifically in gWAT (VAT) but essentially no expression in the other tissues analysed here, and after subcutaneous expression may as found in one case result in expression in subcutaneous white adipose tissue (scWAT, SCAT) and some expression in BAT, and specifically predominantly in expression in ingWAT.

[0046] Fig. 6 shows results for transduction specificity for the vector AAVl-VP48-CMV-ZsGreenl by expression of ZsGreenl in relation to expression of this reporter in liver anterior lobe. Like transduction strength, transduction specificity after intravenous administration is essentially absent, after intraperitoneal administration very specific for gWAT, and after subcutaneous administration in one case high expression in white adipose tissue (scWAT), showing also some specificity for BAT, and specifically predominantly for ingWAT. Fig. 7 shows results for transduction strength of AAV1 with the insert VP51 of the invention and containing the same expression cassette for ZsGreenl (AAVl-VP51-CMV-ZsGreenl), in relation to expression of ZsGreenl as transduced by comparative AAV8-CMV-ZsGreenl. These results show that this AAV with insert VP51 after intravenous administration only in anterior lobe and posterior lobe results in low expression of the reporter, after intraperitoneal administration results in high expression specifically in gWAT but essentially no expression in the other tissues analysed here, and after subcutaneous administration some low expression in liver median lobe and left lobe, and high expression in BAT and in ingWAT only.

[0047] Fig. 8 shows results for transduction specificity for the vector AAVl-VP51-CMV-ZsGreenl by expression of ZsGreenl in relation to expression of this reporter in liver anterior lobe. Unlike transduction strength, transduction specificity after intravenous administration is similar to liver anterior lobe also in liver posterior lobe, in liver median lobe and liver left lobe, and also in BAT, and very low in scWAT. After intraperitoneal administration expression is similarly specific for liver anterior lobe and posterior lobe, in liver median lobe and liver left lobe, and high for gWAT and lower for ingWAT. After subcutaneous administration, specificity is similar in liver anterior lobe and posterior lobe as well as in gWAT, and very high in liver median lobe, in scWAT, in AT and ingWAT.

[0048] Fig. 9 shows measurement results for tropism for liver of AAVl-VP48-CMV-ZsGreenl and of AAVl-VP51-CMV-ZsGreenl of the invention and for comparative AAV8-CMV- ZsGreenl for different routes of administration. After intravenous administration (IV RoA), the viral vectors of the invention have a much lower tropism for the liver lobes analysed than comparative AAV8-CMV-ZsGreenl. After intraperitoneal administration (IP RoA), the viral vectors of the invention have essentially no tropism for the liver lobes analysed compared to expression of the reporter from transduction by AAV8-CMV-ZsGreenl.

[0049] These in vivo results show that the viral vectors of the invention are specific for adipose tissue, show little to no off-target transduction, e.g. little to no transduction of liver tissue compared to AAV8, and that the viral vectors of the invention are effective in delivering and expressing a nucleic acid construct by transduction of tissue in a mammal, depending on the route of administration. Fig. 10 results for quality control of AAVl-VP48-CMV-ZsGreenl and of AAV1-VP51- CMV-ZsGreenl of the invention and for comparative AAV8-CMV-ZsGreenl of viral vector production in HEK293 producer cells. Titers were determined after purification of producer cell medium supernatant by affinity chromatography on an AKTA column, titers were determined by mass spectrometry (Refeyn SamuxMP). The titers of viral particles for the viral vectors of the invention as well as of the comparative vector are in the same range, i.e. approx. 3 x 1013vg / mL, indicating that the insert into VP1 according to the invention does not have a significant negative influence on viral vector production.

Claims

Claims1. AAV vector for use in transducing adipocytes residing in adipose tissue, wherein the viral vector contains VP1 of AAV1 and in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 1 to SEQ ID NO: 80.

2. AAV vector for use according to claim 1 for use in transducing adipocytes residing in adipose tissue by oral administration, wherein the viral vector in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 41 to SEQ ID NO: 80.

3. AAV vector for use according to claim 2, for use in transduction of adipocytes residing in SCAT, wherein the viral vector in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 41 to SEQ ID NO: 60.

4. AAV vector for use according to claim 2, for use in transduction of adipocytes residing in VAT, wherein the viral vector in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 61 to SEQ ID NO: 80.

5. AAV vector for use according to claim 1 for use in transfecting adipocytes residing in adipose tissue by intraperitoneal administration, wherein the viral vector in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 1 to SEQ ID NO: 40.

6. AAV vector for use according to claim 5, for use in transduction of adipocytes residing in SCAT, wherein the viral vector in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 1 to SEQ ID NO: 20.

7. AAV vector for use according to claim 6, for use in transduction of adipocytes residing in VAT, wherein the viral vector in its VP1 between amino acid 590 andamino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 21 to SEQ ID NO: 40.

8. AAV vector for use according to one of the preceding claims, wherein the viral vector comprises a VP1 having an amino acid sequence of SEQ ID NO: 82, wherein the amino acids No. 594 to 600 are selected from SEQ ID NO: 1 to SEQ ID NO: 80.

9. AAV vector for use according to one of the preceding claims for use in medical treatment, wherein the viral vector contains a nucleic acid construct encoding an siRNA, a therapeutically active nucleic acid, antibodies, signal molecules, messenger molecules, hormones, genome / epigenome editing tools or providing a template for genome editing.

10. AAV vector for use according to one of the preceding claims for use in the treatment of a genetic defect, cancer or infectious diseases, wherein the viral vector contains a nucleic acid construct containing an expression cassette encoding a protein substituting a genetic defect.

11. AAV vector for use according to one of the preceding claims, wherein the AAV vector is AAV serotype 1 (AAV1).

12. AAV vector containing the VP1 of AAV1 and in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence containing an insert of one of SEQ ID NO: 1 to SEQ ID NO: 80.

13. AAV vector according to claim 12, characterized in that it is contained in a pharmaceutical formulation for oral administration, wherein the viral vector in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 41 to SEQ ID NO: 80.

14. AAV vector according to claim 12, characterized in that it is contained in a pharmaceutical formulation for intraperitoneal administration, wherein the viral vector in its VP1 between amino acid 590 and amino acid 591 of its wild-type sequence contains an insert of one of SEQ ID NO: 1 to SEQ ID NO: 40.

15. Method for transducing adipocytes, comprising providing adipocytes from adipose tissue and contacting the adipocytes in vitro with an AAV vector according to one of claims 12 to 14.

16. Method for treatment of a patient, comprising administration of an AAV vector according to one of claims 12 to 14, for transducing a nucleic acid construct contained in the AAV vector into adipose tissue.

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