Patent Application
20070172460
The present invention relates to a method of producing a repertoire of random peptides on the surface of AAV particles wherein said random peptides are expressed as a fusion with an AAV capsid protein of an AAV particle which displays at its surface said random polypeptides. The invention also relates to a peptide library obtainable by said method as wells as a method of selecting a gene therapy vector specific for a desired cell type comprising the steps of (a) infecting the desired cell type with a AAV display peptide library of the invention and (b) harvesting AAV library particles from the supernatant and/or cell lysates. Finally, the present invention provides AAV vectors obtained by said method which are useful for gene therapy, e.g., AAV vectors targeting preferably primary human coronary artery endothelial cells which are suitable for the treatment of diseases associated with a dysfunction of said cells.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.
Safety and efficacy of human gene therapy continue to be the subject of considerable debate. Problems of current vectors include unintended transduction of certain tissues, adverse immune reactions, and lack of efficient transduction of the tissue of interest. The most commonly used gene transfer systems to date are derivatives of viruses. Many safety and efficacy concerns may be overcome by ablating the endogenous unspecific tropism of the vector and retargeting it to a specific tissue.
To target vectors to cell type specific receptors, ligands must be linked to the vector capsid through chemical (bispecific conjugates) or recombinant (genetically modified capsids) methods. Random phage display peptide libraries can be used to identify ligands binding to certain cell types in vitro or homing to tissue-specific endothelial receptors after intravenous injection in vivo. Such ligands have been used for therapeutic targeting in experimental models. Redirecting viral vectors by means of bispecific molecular conjugates that contain targeting peptides has substantial drawbacks for systemic treatments. These include the lack of stability of the adaptor-vector complex in vivo and immunogenicity of the adaptor molecule itself. Hence, retargeting gene therapy vectors by incorporating ligands directly into their viral capsid is preferable. On the other hand, incorporating peptides selected by phage display directly into the viral capsid can be successful but also presents limitations. The conformation of peptides in the virus protein context may be altered and the ligand-receptor binding specificity and affinity may be decreased or lost. Furthermore, a peptide isolated by phage display may not function efficiently for gene therapy vector targeting purposes if the respective receptor does not internalize the ligand or internalizes it in a manner not supporting transgene expression.
Thus, there is a need for targeted gene therapy AAV vectors, that overcome the limitations and disadvantages of the approaches of the prior art.
The present invention relates to a method of producing a repertoire of random peptides on the surface of AAV particles wherein said random peptides are expressed as a fusion with an AAV capsid protein of an AAV particle which displays at its surface said random polypeptides. The invention also relates to a peptide library obtainable by said method as well as a method of selecting a gene therapy vector specific for a desired cell type comprising the steps of (a) infecting the desired cell type with a peptide library of the invention and (b) harvesting AAV library particles from the supernatant and/or cell lysates. Finally, the present invention provides AAV vectors obtained by said method which are useful for gene therapy, e.g., AAV vectors targeting primary human coronary artery endothelial cells which are suitable for the treatment of diseases associated with a dysfunction of said cells.
In view of the need of improved targeted gene therapy AAV vectors, the technical problem underlying the present invention is to provide targeted gene therapy AAV-vectors, that overcome the limitations and disadvantages of the approaches of the prior art.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims. For solving the above identified technical problem a peptide screening system displayed directly on the gene therapy vector itself was designed. As a tool for peptide display recombinant adeno-associated virus type 2 (AAV-2) was chosen. AAV vectors are attractive because they are non-pathogenic, transduce cells independently of proliferation, and achieve long-term transgene expression in vitro and in vivo. Heparan sulfate proteoglycan serves as a primary attachment receptor for AAV-2 (Summerford and Samulski, Journal of Virology 72 (1998), 1438-1445). Human fibroblast growth factor receptor-1 and αVβ5 integrin have been proposed as secondary receptors. The single-stranded AAV genome contains two open reading frames, rep and cap, flanked by inverted terminal repeats (ITRs) containing the cis-elements required for replication and packaging (Samulski et al., Journal of Virology 61 (1987), 3096-3101). The cap gene encodes three capsid proteins, VP1-3 (Beccera et al., Journal of Virology 62 (1988), 2745-2754). Antigenic regions of AAV-2 capsids have been mapped and their role in virus-cell interaction and infection neutralization has been described. Moreover, several sites within the AAV capsid amenable to incorporation of targeting peptides have been identified (Girod et al., Nature Medicine 5 (1999), 1052-1056; Rabinowitz et al., Virology 265 (1999), 274-285; Shi et al., Human Gene Therapy 12 (2001), 1697-1711; Wu et al., Journal of Virology 74 (2000), 8635-8647). Inserting a peptide containing an RGD-sequence into loop IV of VP1-3 leads to retargeting AAV-2 vectors to a poorly transducible integrin-expressing cell line(Girod et al., 1999). The same insertion site has been used to retarget AAV by means of phage-derived peptides (Grifman et al., Molecular Therapy 3 (2001), 964-975; Nicklin et al., Molecular Therapy 4 (2001), 174-181). The structure of the AAV-2 capsid has been resolved recently. These data may form the basis for the rational design of new ligand insertion sites (Xie et al., PNAS USA 99 (2002), 10405-10410.
Building on these studies, a peptide library displayed on the surface of AAV-2 was designed. A random peptide library was cloned into the capsid such that it was exposed on the surface of the vector at a site critical for viral attachment to the target cell, thereby diminishing the non-specific endogenous tropism of the vector. The pool of AAV particles obtained, in which each clone displays a different surface peptide, represents a random AAV display peptide library. Such libraries were used to select for AAV infecting primary human coronary artery endothelial cells. The selected particles displayed shared common peptide motifs attesting to the power of the selection. AAV vectors displaying targeting peptides showed superior transduction of coronary artery endothelial cells compared to control vectors carrying the wild-type capsid. This effect was cell type-specific because it was only observed in endothelial cells and not in HeLa control cells.
The present approach shows that viral gene therapy vectors can be used to display peptide libraries within the steric context of the intact virus capsid. Such technology allows selection for vectors transducing the cell type of interest. The AAV-construct of the present invention permits efficient directional cloning of a random oligonucleotide insert allowing the highest possible diversity of the plasmid library. The library is placed within the capsid behind arginine (R) 588 close to a position in which short peptide sequences have-been successfully introduced to retarget AAV-2. The residue R588 itself is involved in heparin binding of AAV-2 (unpublished observations) and insertions at this site have been shown to affect heparin binding activity of the AAV-2 capsid. This effect was confirmed by the observation of a reduction in heparin binding of the peptide display library as well as of the selected clones.
AAV peptide library production was developed as a three-step process (plasmid level, shuttle level, library level). A diversity of 1.1×108 clones was achieved at the plasmid level. The AAV library transfer shuttle production in the next step was performed by using a vast excess of library plasmids over producer cells. This strategy resulted in more than 1012 shuttle particles with packaged library DNA, thus preserving the initial diversity of 108. The final library was generated by infecting producer cells at an MOI of 1-5 replicative units per cell, resulting in a diversity of approximately 2.5×107 which is limited exclusively by the number of cells used for production. Although it is impossible to determine directly the diversity of the final product, it can be expected that it comprises approximately 107 different clones.
The library production method of the present invention allows packaging of wild-type AAV genomes to some degree (
Preferably, for the production of the primary library (library transfer shuttles) a wild-type AAV-2 genome is used alternatively in which the codon usage of the (complete) cap gene is modified in order to prevent wild-type virus generation by homologous recombination with the library plasmids. In a further improvement of this library production step the wild-type capsid protein is generated by expression via recombinant adenovirus harbouring the codon-modified AAV-2 cap gene.
Independently produced libraries with a random peptide insert were panned on primary human coronary artery endothelial cells. After two rounds of selection, we enriched for clearly distinguishable peptide motifs mediating superior, cell-type directed infection and transduction of target cells compared with untargeted vectors in a range similar to previously reported AAV retargeting strategies (Nicklin et al., 2001). At least three peptide motifs were enriched over subsequent rounds of selection. Two of the motifs, NSVRDLG/S and NSVSSXS/A shared the tripeptide NSV, but were quite different in the other amino acid residues. Enrichment of such prominent peptide motifs after only two rounds of selection is remarkable and suggests (a) high expression of the corresponding receptors on target cells and/or (b) a clear advantage of such receptors over others in binding and/or internalizing the virus as well as allowing for viral gene expression and replication. It is important to note that almost all of the amino acid residues being part of the consensus motifs were at largely invariable positions within the peptide insert. This may indicate that the surrounding capsid protein conformation takes part in the binding of the selected clones to the target cell receptors. This fact supports our hypothesis that the selection of targeting peptides for gene therapy vectors is accomplished most appropriately by taking the capsid protein context into account during the screening process as it may be a fixed unit in conjunction with the library peptide that takes part in the targeting process.
To conclude, the above findings open new perspectives in the field of gene therapy. Any cell type of interest may be used to select for AAV vectors with altered tropism. Further development of the system will also allow for selection of AAV in vivo rendering vectors which specifically transduce certain tissues such as heart, lung or tumors. The novel vectors binding to primary human coronary artery endothelial cells described here may be applicable in clinical settings to treat human coronary heart disease. Beyond that, and perhaps more importantly, the novel technology of specific vector selection on any cell type or tissue of interest may have large potential in the development of targeted gene therapy in general.
To summarize, this is the first demonstration of a peptide display system on vector capsids of potential gene therapy vectors. The vector targeting system of the present invention has substantial advantages over other combinatorial approaches such as phage display and may have a broad range of applications in biotechnology and medicine. The present findings allow the selection of specific vector capsids to target gene therapy potentially to any given cell type of interest.
In a first embodiment, the present invention relates to a method of producing a repertoire of random peptides on the surface of AAV particles, which method comprises: expressing in a recombinant host cell nucleic acids encoding a diverse population of peptides, wherein each peptide is expressed as a fusion with an AAV capsid protein of an AAV particle which displays at its surface said diverse population of peptides, said AAV particle having the ability to replicate provided by genetic information packaged therewith and a helper vector providing the adenovirus helper functions.
Unless otherwise stated, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H. G. W., Nagel, B. and Kölbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basle, Switzerland, ISBN 3-906 390-13-6. The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a mammal, preferably a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
As used herein, the term “vector providing the adenovirus helper functions” relates to a vector, preferably a plasmid, which is used to complement functions which are needed for AAV replication and packaging. Examples of suitable helper vectors are shown in Example 1(A),(D).
As used herein, the term “peptide” preferably means peptides having a length of 4 to 30 amino acids. As used herein, the term “repertoire of random peptides” relates to peptides having at least 104 different sequences. The random amino acid sequence is preferably encoded by random oligonucleotides with each random position being encoded by NNK or NNB codons.
Based on the experiments of the examples, below, the person skilled in the art can construct nucleic acid molecules encoding said peptides as AAV capsid fusion constructs which are presented on the AAV capsid surface in such a way that targeting of a desired cell type can be achieved. For the manipulation in prokaryotic cells by means of genetic engineering said nucleic acid molecules or parts of these sequences can be introduced into plasmids allowing a mutagenesis or a modification of a sequence by recombination of DNA sequences. By means of conventional methods (cf. Sambrook and Russel, 2001, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, NY, USA) bases can be exchanged and natural or synthetic sequences can be added. In order to link the DNA fragments with each other adapters or linkers can be added to the fragments. Furthermore, manipulations can be performed that provide suitable cleavage sites or that remove superfluous DNA or cleavage sites. If insertions, deletions or substitutions are desired, in vitro mutagenesis, primer repair, restriction or ligation can be performed. As analysis methods usually sequence analysis, restriction analysis and other biochemical or molecular biological methods are used.
Methods for generating a pool of random oligonucleotides encoding a repertoire of peptides useful for the present invention are well known in the art, see, e.g Smith, G. P. and Scott J. K., Meth. Enzymol. 1993, 217: 228-257 and Example 1(C), below.
AAV vectors suitable for use in the present invention comprise at least the AAV-2 cap gene. AAV vectors suitable for use in the present invention are based on AAV 1 -8, with AAV-2 being preferred.
Any capsid protein of AAV is suitable for insertion of the peptides of the invention as long as their presentation on the AAV surface is not hampered. A preferred capsid protein is VP3. The preferred site of insertion is at or close to the top of the threefold spikes on the AAV-2 capsid surface with the most preferred site of insertion being at nucleotide position 3967 (behind arginine 588).
Preferably, the AAV vector is manipulated in such a way around the site for insertion of the foreign peptide that in case that no insertion by a suitable oligonucleotide occurs the reading frame is shifted by one nucleotide preventing generation of AAV vectors without an insert, as, e.g., illustrated in
In a further embodiment, the present invention relates to a method of producing a repertoire of random peptides on the surface of AAV comprising the following steps:
In preferred embodiments of the methods of the invention, the adenovirus in step (c) is Ad5. Alternatively, any virus or genomes my be used which provide full helper functions for AAV, e.g. Ad2, HSV-1 or baculovirus.
Alternatively, a repertoire of random peptides on the surface of AAV capsids can be produced by the following steps: (a) expression in a producer host cell (i) nucleic acids encoding a diverse population of peptides, wherein each peptide is expressed as a fusion with an AAV capsid protein of an AAV particle which displays at its surface said diverse population of peptides, said AAV particle having the ability to replicate provided by genetic information packaged therewith by transfecting the nucleic acids at a concentration of approximately one plasmid per cell and (ii) a helper vector providing the adenovirus helper functions, (b) harvesting and purifying the AAV capsids obtained in step (a).
The host cells useful in the methods of the invention are not critical and comprise mammalian cells, preferably human cells such as, e.g., 911 cells, 293 cells, Hela cells, CHO cells, with 293T cells being particularly preferred.
The present invention also relates to a peptide library comprising a repertoire of random peptides on the surface of AAV particles, preferably,AAV-2 particles which is obtainable by a method of the invention.
The present invention also relates to a method of selecting a gene therapy vector specific for a desired cell type, comprising the following steps:
Alternatively to the infection with an adenovirus, oligonucleotide inserts encoding the peptide ligands of successfully infecting AAV library particles are PCR amplified and recloned into the Sfi I site of the library plasmid pMT 187-0-3, packaged into AAV library particles as described above and used for the next round of selection.
Methods for infecting cells with AAV vectors, harvesting and purifying AAV particles (peptide library biopanning) are well known to the person skilled in the art and, e.g., described in Example 1(E), below.
After several rounds of infection and selection, viral DNA is extracted from cells productively infected with enriched AAV library particles. The oligonucleotide inserts encoding the peptide ligands are amplified by PCR and subcloned into an AAV “ITR-less” helper plasmid which is used for production of AAV vectors according to standard techniques.
By use of the methods of the invention AAV vectors targeted to any desired cell type can be selected. A preferred cell type are human coronary artery endothelial cells.
In a further embodiment, the present invention relates to a an AAV vector targeting a primary human coronary artery endothelial cell, which is obtainable by the selection method of the invention or an AAV vector targeting a primary human coronary artery endothelial cell expressing a peptide as a fusion with a capsid protein, wherein said capsid protein comprises an amino acid sequence selected from the group of amino acid sequences consisting of the amino acid sequences shown in Table 3 or 4.
Finally, the present invention relates to a method of treatment using an AAV vector selected according to a method of the present invention, e.g., an AAV vector suitable for the treatment of a disease associated with a dysfunction of primary human coronary artery endothelial cells comprising administering to a mammalian subject a therapeutically effective amount of a gene therapy AAV vector containing in its capsid protein a peptide of the invention. The invention may be applied for any disease treatable with targeted AAV vectors, e.g. infectious diseases, cardiovascular diseases or coronary heart diseases.
The pharmaceutical composition containing an AAV vector of the invention, further, contains a pharmaceutically acceptable excipient, diluent or carrier. Examples of suitable pharmaceutical carriers etc. are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration. The route of administration, of course, depends, inter alia, on the kind of vector contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind and stage of infection or disease, general health and other drugs being administered concurrently.
The following Examples illustrate the invention.
(A) Cell culture, transfection, virus production and titering.
Human coronary artery endothelial cells were maintained in the medium supplied by the cell provider (Promocell; Heidelberg, Germany). 293T and HeLa cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS). Transfections were performed by calcium phosphate precipitation (Hauswirth et al., Methods in Enzymology 316 (2000), 743-761). For production of AAV, we transfected 293T cells with the pTAV (Heilbronn et al., Virology 64 (1990), 3012-3018) or pSub201(Samulski et al., Journal of Virology 61 (1987), 3096-3101) plasmid, or their mutant derivatives, along with pXX6 (Xiao et al., Journal of Virology 72 (1998), 2224-2232) or pDG6VP (Dubielzig et al., Journal of Virology 73 (1999), 8989-8998), containing the adenovirus helper functions. After 72 h cells were harvested and viruses were purified by using iodixanol gradients (Hauswirth et al., 2000). Wild-type Ad5 was used for library particle amplification and inactivated thereafter at 55 ° C. for 30 min. The AAV genomic, capsid, and replicative titers were determined as described (Grimm et al., Gene Therapy 6 (1999), 1322-1330). For heparin binding, 109-1010 particles in PBS containing 1 mM MgCl2 and 2.5 mM KCl (PBS-MK) were bound to 1 mL heparin agarose (Sigma; St. Louis, Mo.), washed twice with 5 mL PBS-MK, and eluted twice with 2 mL PBS containing 1 M NaCl. Fractions were collected and analyzed with the A20-enzyme-linked immuno assay (ELISA)(Grimm et al., 1999).
(B) AAV library backbone plasmid
The plasmid serving as a backbone for cloning of the random oligonucleotides was developed from pSub201 (Samulski et al., 1987), containing the wild-type AAV genome. At nucleotide position 3967, two incompatible (to prevent autoligation after digestion) SfiI restriction sites were generated (
(C) Generation of random libraries on the plasmid level
The degenerate oligonucleotide insert was designed to display two different BglI restriction sites on each end allowing for directional in-frame cloning into the SfiI digested pMT187-0-3 plasmid. The following degenerate oligonucleotide encoding a random seven residue peptide insert at position 3967 in the AAV genome was synthesized (University of Freiburg Oligonucleotide Synthesis Core Facility): 5′-CAGTCGGCCAGAGAGGC (NNK) 7GCCCAGGCGGCTGACGAG-3′. Second strand synthesis was done by using the sequenase enzyme from the Sequenase Kit (Amersham; Freiburg, Germany) and the second strand primer 5′-CTCGTCAGCCGCCTGG-3′. The double stranded insert was purified using the QIAquick Nucleotide Removal Kit (Qiagen; Hilden, Germany). The 15 bp stuffer within pMT187-0-3 was cleaved by SfiI digestion and the plasmid was purified using the QIAquick PCR Purification Kit (Qiagen). Plasmid backbone and insert were ligated in a 1:15 molar ratio. Ligated plasmids were transformed into electrocompetent DH5α bacteria using the Gene Pulser (Biorad; Munich, Germany). Plasmid library diversity was determined by the number of clones growing from a representative aliquot of the transformed bacteria on agar plates containing 150 μg/mL ampicillin. Transformed bacteria were grown to saturation and the library plasmids were purified using Qiagen's Plasmid Prep Kit. 20 randomly picked clones from the plasmid library were sequenced to verify the presence of the random insert.
(D) Generation of AAV library transfer shuttles and the random AAV display peptide library
The AAV display peptide library was made from plasmids in a two-step system (
(E) AAV peptide library biopanning
1.2×106 primary human coronary artery endothelial cells were infected at 70% confluency with the AAV display peptide library at an MOI of 100 or 400 capsids/cell, respectively. After 6 h cells were washed with PBS followed by incubation with Ad5 at an MOI of 10 pfu/cell until observing 50% cytopathic effect. Replicated AAV particles were harvested from supernatant and from cell lysates and titered. For each subsequent selection round, preselected AAV library particles were reapplied to the target cells at an MOI of 100 to 1000 capsids/cell.
(F) PCR and Sequencing of AAV library clones
DNA extracted from AAV by the QIAamp Tissue Kit (Qiagen) served as template for a PCR with the primers 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ and 5′-TGATGAGAATCTGTGGAGGAG-3′. PCR products were analyzed by gel electrophoresis, digested with BglI and cloned into the SfiI-digested pMT187-0-3 plasmid. Randomly assigned clones were sequenced using the primer 5′-GTGTGTTGACATCTGCGGTAGCTGGTGCTGTGTTGCCTCTCTGGAGGTTGGTAG-3′.
(G) Single AAV clone replication assay
AAV clones harboring modified capsids were generated using pMT187-0-3 plasmid containing PCR amplified inserts (c.f. F) and titered. Replicative titers were determined in primary coronary artery endothelial cells grown in 96-well plates. 3000 cells/well were incubated with serial dilutions of AAV clones, superinfected with Ad5 at an MOI of 10 pfu/cell and processed as described (Grimm et al., 1999).
(H) Single AAV clone gene transfer assay
To obtain rAAV-vectors carrying a luciferase reporter gene, the modified capsid region was excised by digestion with XcmI/BsiWI from plasmids of selected AAV clones and inserted into pBSΔTR18 (Weger et al., 1997), providing rep- and cap-genes without ITRs. 293T cells were cotransfected with the modified pBSΔTR18, pDGΔVP, and pUF2CMV-Luc, a derivative of pUF2 (Zolotukhin et al., 1996) harboring the luciferase gene from pGL3-basic (Promega, Mannheim, Germany). 3000 coronary endothelial cells per well and 5000 HeLa cells per well were seeded in 96-well plates and incubated with AAV luciferase vectors at an MOI of 104 capsids/cell. After four days, cells were harvested and reporter gene activity was determined by the firefly luciferase assay (Promega).
An AAV-2 library based on the pMT187-0-3 backbone plasmid was designed to display a peptide with seven random amino acid residues within the VP capsid protein domain required for binding of AAV-2 to its natural receptor heparan sulfate (Wu et al., 2000; Xie et al., 2002); (
After establishing the plasmid library, the AAV particle library production was performed in two additional steps (
The genomic, capsid, and replicalive titers were determined as described (Grimm et al., 1999).
atwo shuttle preparations were made independently: 2/02 and 3/02.
btwo library preparations were made independently, one derived from shuttle preparation 2/02 (2/02-Lib) and one from shuttle preparation 3/02 (3/02-Lib)
Heparin binding of wild-type controls was 100%.
athe low capsid titer of the library did not allow for conduction of a heparin binding assay.
293T cells were infected with AAV library transfer shuttles at a multiplicity of infection (MOI) of 1 and 5 replicative units per cell. This way, we estimated uptake and propagation of approximately 1-5 library genomes per cell. Cells were superinfected with wild-type adenovirus type 5 (Ad5), resulting in efficient production of a random AAV-2 display peptide library (Table 1). Heparin binding analyzed in one library preparation was reduced to 36% of wild-type AAV-2 (Table 2). These results indicate that it is possible to produce AAV libraries efficiently in a two-step system from AAV- encoding plasmids containing random oligonucleotide inserts.
Selection of AAV library clones targeting primary human coronary artery endothelial cells Primary human endothelial cells are poorly susceptible to wild-type AAV-2 transduction (Nicklin et al., 2001; Pajusola et al., Journal of Virology 76 (2002), 11530-11540). To select for AAV capsids that allow higher efficiency transduction of these cells, 1.2×106 primary coronary artery endothelial cells were infected with the AAV library at an MOI of 100-400 capsids per cell. The low replicative titer of the initial libraries on coronary endothelial cells (103/mL for 3/02-Lib and 104/mL for 3/02-Lib) indicated a low number of clones within the AAV library displaying peptides that allow AAV binding and entry into coronary endothelial cells. Thus, we reasoned that the actual infection rate should not exceed one particle/cell in this setting. Cells were superinfected with Ad5, allowing for amplification of internalized AAV library clones. Amplified AAV were recovered and subjected to two more rounds of selection to enrich for AAV particles that bind to, are internalized by, and replicate within coronary artery endothelial cells. The DNA region containing the oligonucleotide insert of AAV particles recovered from all the stages of selection was amplified by PCR (
The NSVRDLS/G consensus motif is shown in blue, the enrichment of PRSVTVP is highlighted in orange.
aMOI used for selection based on capsid titer.
The NSVSSXS/A consensus motif is shown in green. The MOI used for selection was 100 capsid units cell.
Stocks of several AAV clones displaying a targeting peptide were produced and evaluated as follows: Analysis of heparin binding of five representative clones revealed a reduction to 1-11% relative to wild-type (Table 2). Infection and replication efficiency in primary coronary artery endothelial cells of targeted clones was compared to wild-type AAV-2 and a randomly selected control clone (LPQARSH) of an unselected AAV library. All selected clones showed replicative titers 10- to 630-fold higher than those of wild-type AAV-2 (
To validate the targeting effect of the selected peptides for AAV-mediated gene transfer, endothelial cells were transduced with targeted and untargeted vectors harboring a luciferase reporter gene. Gene transfer was enhanced 4- to 40-fold compared to AAV vectors carrying wild-type capsids (
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/02923 | 3/19/2004 | WO | 1/12/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60455594 | Mar 2003 | US |
