Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses.

We have described the development of capsid-modified next-generation AAV vectors for both AAV2 and AAV3 serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses. We have also described the development of genome-modified AAV vectors, in which the transcriptionally inactive, single-stranded AAV genome was modified to achieve improved transgene expression. Here, we describe that combination of capsid modifications and genome modifications leads to the generation of optimized AAV serotype vectors, which transduce cells and tissues more efficiently, both in vitro and in vivo, at ∼20-30-fold reduced doses. These studies have significant implications in the potential use of the optimized AAV serotype vectors in human gene therapy.

Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo.

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of recombinant adeno-associated virus 2 (AAV2) vectors, which negatively impacts the transduction efficiency of these vectors. Because ubiquitination occurs on lysine (K) residues, we performed site-directed mutagenesis where we replaced each of 10 surface-exposed K residues (K258, K490, K507, K527, K532, K544, K549, K556, K665, and K706) with glutamic acid (E) because of similarity of size and lack of recognition by modifying enzymes. The transduction efficiency of K490E, K544E, K549E, and K556E scAAV2 vectors increased in HeLa cells in vitro up to 5-fold compared with wild-type (WT) AAV2 vectors, with the K556E mutant being the most efficient. Intravenous delivery of WT and K-mutant ssAAV2 vectors further corroborated these results in murine hepatocytes in vivo. Because AAV8 vectors transduce murine hepatocytes exceedingly well, and because some of the surface-exposed K residues are conserved between these serotypes, we generated and tested two single mutants (K547E and K569E), and one double-mutant (K547 + 569E) AAV8 vector. However, no significant increase in the transduction efficiency of any of these mutant AAV8 vectors was observed in murine hepatocytes in vivo. These studies suggest that although targeting the surface-exposed K residues is yet another strategy to improve the transduction efficiency of AAV vectors, phenotypic outcome is serotype specific.

Enhanced transgene expression from recombinant single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and in murine hepatocytes in vivo.

UNLABELLED: We have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573-580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668-1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy. IMPORTANCE: The results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy.

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo.

BACKGROUND AIMS: Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. METHODS: We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. RESULTS: We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. CONCLUSIONS: These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

High-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo. However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells. In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

A simple method to increase the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo.

We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors.

Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina.

Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.

High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.

Human hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus serotype 3.

Adeno-associated viruses (AAVs) use a variety of cellular receptors/coreceptors to gain entry into cells. A number of AAV serotypes are now available, and the cognate receptors/coreceptors for only a handful of those have been identified thus far. Of the 10 commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells in general. However, in our more recent studies, we observed that AAV3 vectors transduced human liver cancer cells remarkably well, which led to the hypothesis that AAV3 uses hepatocyte growth factor receptor (HGFR) as a cellular coreceptor for viral entry. AAV3 infection of human liver cancer cell lines was strongly inhibited by hepatocyte growth factor, HGFR-specific small interfering RNA, and anti-HGFR antibody, which corroborated this hypothesis. However, AAV3 vectors failed to transduce murine hepatocytes, both in vitro and in vivo, suggesting that AAV3 specifically uses human HGFR, but not murine HGFR, as a cellular coreceptor for transduction. AAV3 may prove to be a useful vector for targeting human liver cancers for the potential gene therapy.

High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.

Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is ∼10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by ∼130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80-90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors.

Optimized adeno-associated virus (AAV)-protein phosphatase-5 helper viruses for efficient liver transduction by single-stranded AAV vectors: therapeutic expression of factor IX at reduced vector doses.

Abstract Our studies have shown that coinjection of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors carrying the enhanced green fluorescent protein (EGFP) gene with self-complementary (sc) AAV2-T cell protein tyrosine phosphatase (TC-PTP) and scAAV2-protein phosphatase-5 (PP5) vectors resulted in an approximately 16-fold increase in EGFP expression in primary murine hepatocytes in vivo [Jayandharan, G.R., Zhong, L., Li, B., Kachniarz, B., and Srivastava, A. (2008). Gene Ther. 15, 1287-1293]. In the present studies, this strategy was further optimized to achieve transgene expression at reduced vector/helper virus doses. These included the use of scAAV helper viruses containing (1) hepatocyte-specific promoters, (2) tyrosine-mutant AAV2 capsids, and (3) additional AAV serotype vectors known to efficiently transduce hepatocytes. The hepatocyte-specific transthyretin (TTR) promoter was approximately 6- to 7-fold more efficient than the Rous sarcoma virus (RSV) promoter; tyrosine-mutant AAV2 capsids were approximately 6- to 11-fold more efficient than the wild-type AAV2 capsids; and the AAV8 serotype helper virus was approximately 16-fold more efficient than AAV2 serotype helper virus. With these modifications, the vector dose of the helper virus could be further reduced by approximately 50-fold. Last, coadministration of scAAV8-PP5 helper virus increased coagulation factor IX expression from an ssAAV2 vector by approximately 7- to 10-fold, thereby achieving therapeutic levels at lower vector doses. No adverse effect on hepatocytes was observed under any of these experimental conditions. The strategy presented here should be adaptable to any ssAAV transgene cassette and, specifically, liver-directed applications of ssAAV2 vectors containing larger genes that cannot be encapsidated in scAAV vectors.

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression.

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by approximately 68% and approximately 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.

Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells approximately 10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an approximately 10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.

A dual role of EGFR protein tyrosine kinase signaling in ubiquitination of AAV2 capsids and viral second-strand DNA synthesis.

A 52 kd cellular protein, FK506-binding protein (FKBP52), phosphorylated at tyrosine residues by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), inhibits adeno-associated virus 2 (AAV2) second-strand DNA synthesis and transgene expression. FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and TC-PTP over-expression leads to improved viral second-strand DNA synthesis and improved transgene expression. In these studies, we observed that perturbation of EGFR-PTK signaling by a specific inhibitor, Tyrphostin 23 (Tyr23), augmented the transduction efficiency of the single-stranded AAV (ssAAV) vector as well as the self-complementary AAV (scAAV) vector. Similarly, tyrosine-dephosphorylation of FKBP52 by TC-PTP resulted in increased transduction by both vectors. These data suggested that EGFR-PTK signaling also affects aspects of AAV transduction other than viral second-strand DNA synthesis. We document that inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsids which, in turn, facilitates nuclear transport by limiting proteasome-mediated degradation of AAV vectors. We also document that Tyr23-mediated increase in AAV2 transduction efficiency is not further enhanced by a specific proteasome inhibitor, MG132. Thus, EGFR-PTK signaling modulates ubiquitin (Ub)/proteasome pathway-mediated intracellular trafficking as well as FKBP52-mediated second-strand DNA synthesis of AAV2 vectors. This has implications in the optimal use of AAV vectors in gene therapy.

Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity.

Self-complementary adeno-associated viral (scAAV) vectors bypass the requirement for viral second-strand DNA synthesis, but the packaging capacity of these vectors ( approximately 2.4 kb) is significantly smaller than that of conventional AAV vectors ( approximately 4.8 kb). We constructed human recombinant green fluorescent protein (hrGFP) expression cassettes ranging from 2.3 to 4.1 kb. Each vector was biologically active, but the transduction efficiency of vectors containing <3.3-kb genomes was significantly higher than those containing 3.5-kb genomes or larger. However, scAAV vectors containing up to approximately 3.3-kb genomes also contained single-stranded genomes, and 3.5-kb and larger genomes were packaged only as single-stranded DNA. These data suggest that the maximum packaging capacity of scAAV vectors is approximately 3.3 kb. The production of single-stranded genomes was not due to repair of the terminal resolution site (trs) in the inverted terminal repeats in the AAV genome, but rather was partly due to the use of AAV helper plasmid, known to lead to higher levels of expression of Rep proteins. The use of a helper plasmid known to lead to reduced levels of Rep proteins led to the generation of scAAV vectors that contained approximately 90% of the viral genomes in double-stranded forms. These studies demonstrate the feasibility of achieving encapsidation of larger genomes into scAAV vectors than was suggested originally, but underscore the need to exercise caution in using the appropriate helper plasmid to generate scAAV stocks capable of high-efficiency transduction that are relatively free of single-stranded DNA-containing vectors.

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

Pneumocystis jiroveci in Portuguese immunocompromised patients: association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.

We analyzed the genetic variation among isolates of Pneumocystis jiroveci from Portuguese immunocompromised patients with PCP at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon and at the dihydropteroate synthase (DHPS) gene. Pulmonary secretions from 42 patients with PCP corresponding to 43 episodes were studied. Demographic, immunological, and clinical data were obtained from all patients. By combining the two regions ITS1 and ITS2, we found 17 different ITS types of P. jiroveci, two of them were new types (Pb and Pe). The four most prevalent ITS types were Eg (23.3%), Eb and Ne (11.6% each), and Bi (9.3%). A single type was detected in 95.3% of the samples and 4.7% had mixed infections with three different ITS types. DHPS mutants were present in 17 (46%), and the wildtype was present in 20 (54%) of 37 isolates. No association was found between ITS and DHPS types and between DHPS types and therapy or response to anti-PCP treatment. Type Ne presented an association with negative response to anti-PCP treatment (P<0.001) and with death before 120 days after PCP diagnosis (P=0.025). Type Eb was significantly more common in children than in adults (P=0.001). Our data suggest an association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.