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Evolved properties of Adeno-Associated Virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Previous efforts to re-engineer these properties have focused on variable regions near AAV’s 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of large, structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. There was strong positional, domain-type, and fitness phenotype dependence of insertion permissibility, which clustered into correlated structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified new engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

Adeno-associated virus (AAV)-based gene therapy is undergoing major expansion into clinical practice, with two treatments currently being granted Food and Drug Administration (FDA) approval. However, the presence of pre-existing neutralizing antibodies (NAB) is one of the significant hurdles for the clinical application of AAV vectors that significantly limits the patient population, which benefits from the treatment. A reliable diagnostic to evaluate the patient's seropositivity is required to ensure the effectiveness of the AAV-mediated therapeutic. Here, we describe a simple method for the determination of AAV NAB activity based on our finding that Compound C makes HEK293 cell highly permissive for infection by 10 commonly used AAV serotypes.

There are a number of respiratory diseases characterized by the presence of excess neutrophil elastase (NE) activity in tissues, including cystic fibrosis and chronic obstructive pulmonary disease (COPD). NE is considered a primary contributor to disease development, but the precise mechanism has yet to be fully determined. We hypothesized that NE alters the function of macrophages (Mɸ) which play a critical role in many physiological processes in healthy lungs. We demonstrate that monocyte-derived Mɸ exposed to NE releases active matrix metalloproteinases (MMPs), increase expression of pro-inflammatory cytokines TNFα, IL-1β, and IL-8, and reduce capacity to phagocytose bacteria. Changes in Mɸ function following NE treatment were accompanied by increased adhesion and cytoskeleton re-arrangement, indicating the possibility of integrin involvement. To support this observation, we demonstrate that NE induces phosphorylation of kinases from the Src kinase family, a hallmark of integrin signaling activation. Moreover, pretreatment of Mɸ with a specific Src kinase inhibitor, PP2 completely prevents NE-induced pro-inflammatory cytokine production. Taken together these findings indicate that NE participates in lung destruction not only through direct proteolytic degradation of matrix proteins, but also through activation of Mɸ inflammatory and proteolytic functions.

Despite efforts to use adeno-associated viral (AAV) vector–mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to KrasG12D+/−, KrasG12D+/−/Pten+/−, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45–70%) compared with WT-AAV8 (7%; p

Despite the progress made in AAV-based gene therapy targeting different organ systems, lung-targeted gene therapy using AAV vectors has not been effective, mostly due to the poor transduction and un-sustained gene expression in airway epithelium. Furthermore, concerns over possible harmful insertional mutagenesis seen in other cell types, particularly hepatocytes, raised a question about AAV safety. In this study, we evaluate the long-term persistence of this vector in mouse lungs and any possible harmful integration of these vectors into the host genome. AAV6 vectors expressing reporter gene (firefly luciferase) were delivered to the lungs of C57BL/6 mice through intra-tracheal intubation. Despite the large variation among individual animals, most animals had high and sustained luciferase activity with a peak from 2 to 3 weeks post-transduction before a significant decline between 15 and 19 weeks post-transduction. More importantly, even after its decline, most animals maintained detectable luciferase expression for 150 days or more, which was confirmed by post-necropsy qPCR analysis of luciferase gene expression. At the termination point of experiments, an average of one copy of AAV expression cassette per mouse genome was detected. We also found that partial overlaps between the AAV6 expression cassette and the mouse genome were distributed broadly with no apparent systematic preference in any mouse chromosomal map location. In summary, our data suggest that AAV6 mediated long-term gene expression in the lungs with no evidence of genomic integration, and thus, any insertional mutagenesis., Highlights • AAV6 vectors delivered by intra-tracheal intubation were persist in the mice lungs for over 150 days. • Persistent of the gene expression was confirmed by qPCR analysis and was 17-fold higher over mock. • On average, there were approximately 1 copy of AAV sequence per mouse genome. • No “hot spot” was identified that suggest any preference of the AAV integration in the mouse genome.

We recently showed that the vaccine based on optimized AAV6 vector generates high level of antigen (Ag)-specific immune response. However, certain limitations in use of viral vectors as vaccine carriers need to be overcome to ensure its wide use for clinical application. Particularly, the primary injection of AAV lead to generation of neutralizing antibodies (NAB) against the virus capsid, hampering AAV re-administration. To address this limitation, we exploited the ability of AAV to accumulate in exosomes. AAV vectors associated with exosomes (exoAAV) were purified from conditioned media of HEK 293 cells, along with the standard AAV production (stAAV) from the same lysed cells. Here we report several significant observations that highlight the advantages of using exoAAV for development in a prime-boost protocol. First, we confirmed comparable infectivity between stAAV and exoAAV using YFP and luciferase reporter genes. Second, we showed that exoAAV is highly resistant to NAB compared to stAAV. Third, we developed a prime-boost protocol with injections of stAAV followed by exoAAV expressing OVA Ag as a model system. This protocol led to significantly higher levels of OVA-specific CD8+ T cells compared to the single injection method. The therapeutic potential of prime-boost was evaluated in a melanoma B16F10 tumor prevention model in comparison to single injection vaccination. While a single injection was able to significantly delay tumor growth, the prime-boost protocol completely prevented tumor development. In summary, we use exosome-protected AAV to allow for repeated vaccination, and this method produced a superior protective immune response compared to single vaccination.

We recently showed that a vaccine with optimized AAV6 vector generates a superior immune response against strong antigens (Ag) such as Ovalbumin. However, tumor-associated antigens (TAA) are typically non-mutated self-proteins with low immunogenicity. To increase vaccine efficacy and to reduce tumor evasion of immune attack, we developed a multivalent AAV vaccine that targets four previously characterized melanoma TAA: tyrosinase, premelanosome protein gp100, tyrosinase-related protein 1, and dopachrome tautomerase. The vaccine represents a combination of individual AAV-based vaccines specific to unique TAA. The strength of cytotoxic T cell response against each TAA was shown via IFNγ ELISPOT analysis of re-stimulated splenocytes from immunized mice. Additionally, we showed that vaccine-induced Ag-specific antibodies directed B16F10 melanoma cells to complement-dependent lysis. The vaccine’s therapeutic potential was analyzed in prevention and treatment of B16F10 mouse melanoma. The preventative vaccination significantly reduced the number of metastatic tumor nodules in lungs. Since B16F10 melanomas are immunologically resistant tumors, we combined vaccination with aPD-1 and aPD-L1 checkpoint inhibitors for treatment of established solid tumor. The combination of both vaccine and checkpoint inhibitors significantly delayed tumor growth and extended animal survival. In summary, our preliminary data suggest that a multivalent vaccine based on modified AAV6 vectors generates strong immune responses against endogenous self-proteins, and vaccination may have a therapeutic benefit against both metastatic and solid tumor development.

There are a number of diseases characterized by the presence of neutrophil elastase (NE) activity in tissues including cystic fibrosis and alpha-1-antitrypsin deficiency induced lung destruction. It is generally accepted that NE actively contributes to this pathological process, but the precise mechanisms has yet to be determined. We hypothesized that NE activates the macrophages (M□) pro-inflammatory program. We demonstrate that following NE exposure, monocyte-derived M□ release proteolytic activity composed of several matrix metalloproteinases (MMPs) which could contribute to extracellular matrix (ECM) degradation. NE upregulates expression of M□ derived pro-inflammatory cytokines including TNFα, IL-1β, and IL-8. Thus, NE-activated M□ can contribute to tissue destruction through the proteolytic activity of metalloproteinases and by supporting chronic inflammation through expression of pro-inflammatory cytokines. We also demonstrate that NE increases M□ adhesion that is attenuated by antibodies specific to integrin subunits. We show that the effects of NE on M□ can be mediated through an activation of integrin pathways. In support of integrin involvement, we demonstrate that NE activates the Src kinase family, a hallmark of integrin signaling activation. Moreover, pretreatment of macrophages with a specific Src kinase inhibitor, PP2, completely prevents NE-induced inflammatory cytokine production. Taken together these findings indicate that NE has effect on lung destruction that extends beyond direct proteolytic degradation of matrix proteins.

Viral vectors-based gene therapy is an attractive alternative to common anti-cancer treatments. In the present studies, AAV serotype 6 vectors were identified to be particularly effective in the transduction of human prostate (PC3), breast (T47D) and liver (Huh7) cancer cells. Next, we developed chimeric AAV vectors with Arg-Gly-Asp (RGD) peptide incorporated into the viral capsid to enable specific targeting of integrin-overexpressing malignant cells. These AAV6-RGD vectors improved transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing the viral entry into the cells. We also observed that transduction efficiency significantly improved, up to approximately 5-fold, by the mutagenesis of surface-exposed tyrosine and threonine residues involved in the intracellular trafficking of AAV vectors. Therefore, in our study, the AAV6-Y705-731F+T492V vector was identified as the most efficient. The combination of RGD peptide, tyrosine and threonine mutations on the same AAV6 capsid further increased the transduction efficiency, approximately 8-fold in vitro. In addition, we mutated lysine (K531E) to impair the affinity of AAV6 vectors to heparan sulfate proteoglycan. Finally, we showed a significant increase in both specificity and efficiency of AAV6-RGD-Y705-731F+T492V+K531E vectors in a xenograft animal model in vivo. In summary, the approach described here can lead to the development of AAV vectors with selective tropism to human cancer cells.

Alpha-1 antitrypsin (AAT) deficiency-associated emphysema is largely attributed to insufficient inhibition of neutrophil elastase released from neutrophils. Correcting AAT levels using augmentation therapy only slows disease progression, and that suggests a more complex process of lung destruction. Because alveolar macrophages (Mɸ) express AAT, we propose that the expression and intracellular accumulation of mutated Z-AAT (the most common mutation) compromises Mɸ function and contributes to emphysema development. Extracellular matrix (ECM) degradation is a hallmark of emphysema pathology. In this study, Mɸ from individuals with Z-AAT (Z-Mɸ) have greater proteolytic activity on ECM than do normal Mɸ. This abnormal Z-Mɸ activity is not abrogated by supplementation with exogenous AAT and is likely the result of cellular dysfunction induced by intracellular accumulation of Z-AAT. Using pharmacologic inhibitors, we show that several classes of proteases are involved in matrix degradation by Z-Mɸ. Importantly, compared with normal Mɸ, the membrane-bound serine protease, matriptase, is present in Z-Mɸ at higher levels and contributes to their proteolytic activity on ECM. In addition, we identified matrix metalloproteinase (MMP)-14, a membrane-anchored metalloproteinase, as a novel substrate for matriptase, and showed that matriptase regulates the levels of MMP-14 on the cell surface. Thus, high levels of matriptase may contribute to increased ECM degradation by Z-Mɸ, both directly and through MMP-14 activation. In summary, the expression of Z-AAT in Mɸ confers increased proteolytic activity on ECM. This proteolytic activity is not rescued by exogenous AAT supplementation and could thus contribute to augmentation resistance in AAT deficiency-associated emphysema.

Objective Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine (TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus (rAAV) vectors has not been attempted. Methods We synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The protein expression was confirmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into rAAV vectors, under the control of a liver cancer-specific promoter. The liver tumor growth was then monitored following intratumor administration of the rAAV vectors. Results The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin ( TCS ) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of rAAV vectors containing the TCS gene significantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model. Conclusion Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Introduction: Adeno-associated virus (AAV) is a small virus of the parvovirus family and is widely used in clinical-stage gene therapy because of its low toxicity and limited side effects. There are a number of naturally occurring serotypes with different tissue tropism and further engineering of the viral capsid can increase the vectors suitability for use as a cancer vaccine. We recently showed that AAV serotype 6 (AAV6) successfully delivers a transgene to dendritic cells (DC) in vitro. In the present study we analyze several rationally designed AAV6 mutants to more efficiently target DC and further increase an antigen-specific immune response. Methods: AAV6 mutants encoding ovalbumin, tyrosinase, or GFP were developed and produced in the lab by standard procedure. Vaccination was performed as a single intramuscular (i.m.) injection in C57BL/6 mice. The immune response was measured against both ovalbumin (Ova) as a model antigen and tyrosinase (Tyr) as an endogenous antigen. The Ova-specific CD8 T-cells were identified in peripheral blood and splenocytes by binding epitope-specific tetramers. Activity of antigen-specific CD8 cells was estimated by IFNy production measured by ELISpot, and by in vivo and in vitro killing assays. A humoral response was analyzed by the presence of antigen-specific antibodies in mouse serum measured by ELISA or by Western Blotting. Vaccination with GFP was used to show direct antigen expression in DC. Briefly, 3 days after i.m. injection DC from drained lymph nodes were isolated by FACS (CD11c+/MHC II+) and the presence of GFP expression was analyzed by wide-field fluorescence microscopy. Results: Several significant observations were made: (i) optimized AAV6 directly targets DC in vivo as evidenced by the presence of GFP positive DC in mouse drained lymph nodes after i.m. injection with AAV6-GFP; (ii) AAV6 vectors induced a strong immune response not only against a strong immunogen such as Ova but also against a weak endogenous antigen such as Tyr. (iii) Transgene-specific cytotoxic CD8 T- cells showed superior killing capabilities against cancer cells in vitro and in cytotoxic assay in vivo. (iv) AAV6 based vaccine generates high transgene-specific antibodies titers detectable starting 3 weeks after injection. Our data allows us to choose the AAV6 mutant with superior capacity to induce an immune response against a transgene. As a future direction we will optimize the expression cassette to directly rout an antigen into the MHC class II processing and presentation pathway which is known to dramatically increase vaccine potency. The combination of optimized AAV capsid and expression cassette will be used to confirm an ability of our vaccine approach to reduce tumor growth and extend survival in a murine tumor model. In addition, our technology is highly compatible with, and complementary to current cancer treatments, including other immunotherapy approaches such as checkpoint (PD-1/PD-L1) inhibitors. Citation Format: Karina Krotova, Andrew Day, Edward Hinchcliffe, George Aslanidi. Bioengineering AAV6 vector-based vaccine for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1467.

Dendritic cell (DC)-based immunotherapy has recently demonstrated a great potential for clinical applications; however, additional progress in the methods of tumor-specific antigen delivery to DCs is necessary for the further development of anti-tumor vaccines. To this end, a capsid-optimized adeno-associated virus serotype 6 (AAV6-T492V+S663V) vector was developed by site-directed mutagenesis of surface-exposed serine (S) and threonine (T) residues, which have a critical role in intracellular trafficking of AAV vectors. This double-mutant AAV6 vector had ∼ 5-fold greater transduction efficiency in monocyte-derived DCs (moDCs) compared with wild-type (WT)-AAV6 vectors. The increase in the transduction efficiency correlated with the improved nuclear translocation of AAV6-T492V+S663V over that of the WT-AAV6 vector. Additional studies of the CD11c promoter identified critical regulatory elements that fit into the AAV expression cassette and drive EGFP expression in moDCs. Development of a chimeric promoter (chmCD11c) that contains functional modules of CD11c and a Simian virus (SV40) enhancer element dramatically increased the EGFP expression in moDCs. MoDCs transduced by the capsid-optimized AAV6 vector carrying human prostate-specific antigen (hPSA) driven by CBA (AAV6-T492V+S663V-CBA-hPSA) or chmCd11c (AAV6-T492V+S663V-chmCD11c-hPSA) generated specific T-cell clone proliferation and superior cytotoxic T lymphocytes (CTLs) with higher killing capability against human prostate adenocarcinoma cells, LNCaP, compared with WT-AAV6 induced CTLs. Taken together, these studies suggest that optimization of capsid and promoter components of AAV vectors can be a useful approach for efficient targeting of moDCs and may prove to be a promising tool for cancer immunotherapy.

Chen Ling1,2, Yuan Wang1-3, Yuan Lu4, Lina Wang1-3, Giridhara R Jayandharan5, George V Aslanidi1,2, Baozheng Li1,2, Binbin Cheng1,3, Wenqin Ma1,2, Thomas Lentz6, Changquan Ling3, Xiao Xiao6,7, R Jude Samulski6, Nicholas Muzyczka2,8,9 and Arun Srivastava1,2,8-10* 1Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA 2Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA 3Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China 4Department of Orthopedics & Rehabilitative Medicine, University of Florida College of Medicine, Gainesville, FL, USA 5Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India 6Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 7Division of Molecular Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill, NC, USA 8Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, USA 9Genetics Institute, University of Florida College of Medicine, Gainesville, FL, USA 10Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL, USA *Corresponding author: Arun Srivastava, Division of Cellular & Molecular Therapy, Cancer and Genetics Research Complex, 2033 Mowry Road, Room 492-A, Gainesville, FL 32611-3633, USA, Tel: (352) 273-8259; Fax: (352) 273-8342; E-mail: aruns@peds.ufl.edu

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less “pointed” than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.

Phosphorylation of surface-exposed tyrosine residues negatively impacts the transduction efficiency of recombinant AAV2 vectors. Pre-treatment of cells with specific cellular serine/threonine kinase inhibitors also significantly increased the transduction efficiency of AAV2 vectors. We reasoned that site-directed mutagenesis of surface-exposed serine residues might allow the vectors to evade phosphorylation and thus lead to higher transduction efficiency. Each of the 15 surface-exposed serine (S) residues was substituted with valine (V) residues, and the transduction efficiency of three of these mutants, S458V, S492V and S662V, was increased by up to ≈ 20-fold in different cell types. The S662V mutant was efficient in transducing human monocyte-derived dendritic cells (moDCs), a cell type not readily amenable to transduction by the conventional AAV vectors, and did not induce any phenotypic changes in these cells. Recombinant S662V-AAV2 vectors encoding a truncated human telomerase (hTERT) gene were generated and used to stimulate cytotoxic T cells (CTLs) against target cells. S662V-AAV2-hTERT vector-transduced DCs resulted in rapid, specific T-cell clone proliferation and generation of robust CTLs, which led to specific cell lysis of K562 cells. These studies suggest that high-efficiency transduction of moDCs by serine-modified AAV2 vectors is feasible, which supports the potential utility of these vectors for future human DCs vaccine studies.

There are a number of diseases characterized by the presence of neutrophil elastase (NE) activity in tissues. It is generally accepted that presence of NE actively contributes to a pathological process, but the precise mechanism remains to be elucidated. We hypothesize that one NE action is activation of an inflammatory response in macrophages (Mφ). First, we show that in response to free NE exposure, Mφ release profound proteolytic activity composed of several MMPs which could contribute to extracellular matrix (ECM) degradation. Second, NE treatment also upregulated inflammatory cytokine expression, such as that of TNFα, IL-6, IL-1b, and IL-8. Therefore, Mφ activated by NE can contribute to tissue destruction on several levels, including release of proteolytic activity and by supporting chronic inflammation through expression of chemokines and cytokines. We also demonstrate that NE increases Mφ adhesion which can be attenuated by antibodies against integrin subunits. In support of our hypothesis of integrin involvement, we show an activation of the Srk kinase family in response to NE, which is a hallmark of integrin signaling pathway activation. Moreover, pretreatment of Mφ with specific Srk kinase inhibitor PP2 completely blocked cytokine production in response to NE. In summary, these data propose the mechanism of macrophage activation by NE and also suggest the inhibition of Srk can be potential therapeutic target to reduce NE-induced inflammation in lungs.

Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P3(2), with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress.

Adeno-associated viruses (AAVs) are being developed as gene therapy vectors, and their efficacy could be improved by a detailed understanding of their viral capsid structures. AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than those of other serotypes, which has resulted in efforts to develop this virus as a gene therapy vector for hemophilia A and familial hypercholesterolemia. Pseudotyping studies show that the differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in their capsid viral protein (VP) amino acids. Towards identifying the structural features underpinning these disparities, we report the crystal structure of the AAV8 viral capsid determined to 2.6-Å resolution. The overall topology of its common overlapping VP is similar to that previously reported for the crystal structures of AAV2 and AAV4, with an eight-stranded β-barrel and long loops between the β-strands. The most significant structural differences between AAV8 and AAV2 (the best-characterized serotype) are located on the capsid surface at protrusions surrounding the two-, three-, and fivefold axes at residues reported to control transduction efficiency and antibody recognition for AAV2. In addition, a comparison of the AAV8 and AAV2 capsid surface amino acids showed a reduced distribution of basic charge for AAV8 at the mapped AAV2 heparin sulfate receptor binding region, consistent with an observed non-heparin-binding phenotype for AAV8. Thus, this AAV8 structure provides an additional platform for mutagenesis efforts to characterize AAV capsid regions responsible for differential cellular tropism, transduction, and antigenicity for these promising gene therapy vectors.

The Wnt family of secreted glycoproteins had previously been shown to regulate diverse processes during early development. Wnt signaling also plays a key role in the homeostasis of adult tissues maintaining stem cell pluripotency and determining differentiating cell fate. The age-related decrease in Wnt signaling may contribute to increased muscle adiposity and diminished bone strength. In the current study, we investigated the long-term metabolic consequences of the upregulated Wnt/β-catenin signaling in skeletal muscles of adult diet-induced obese (DIO) rats. To this end, we generated a recombinant adeno-associated virus (rAAV) vector encoding murine Wnt10b cDNA. The long-term expression of rAAV1-Wnt10b was tested after intramuscular injection in the female DIO rat. Animals fed high-fat diet and treated with rAAV1-Wnt10b showed a sustained reduction in body weight compared with controls, and expression of Wnt10b was accompanied by a reduction in hyperinsulinemia and triglyceride plasma levels as well as improved glucose homeostasis. Nuclear magnetic resonance methods revealed that ectopic expression of Wnt10b resulted in a decrease in both global and muscular fat deposits in DIO rats. The long-range effect of locally expressed Wnt10b was also manifested through the increased bone mineral density. The detailed analysis of molecular markers revealed fibroblast growth factor-4 and vascular endothelial growth factor as possible mediators of the systemic effect of Wnt10b transgene expression. Our data demonstrate that altering Wnt/β-catenin signaling in the skeletal muscle of an adult animal invokes moderate responses with favorable metabolic profile, bringing the notion of alternative therapeutic modality in the treatment of obesity, diabetes, and osteoporosis.

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.

In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow-derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (

We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3′ end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA.

Several groups have demonstrated that a number of naturally occurring compounds can significantly enhance the therapeutic efficacy of viral vectors, especially recombinant AAV vectors (Ren et al., Neurosci Lett. 479(3): 187-91, 2010; Zhang et al., Gene Ther. 18(2): 128-34, 2011; Mitchell et al., J Virol. 87(8): 4571-83, 2013; Wang et al., J Integr Med. 12(1): 20-34, 2014). In our recently published studies (Ling et al., Hum Gene Ther. 25(12): 1023-34, 2014), we provided further evidence of enhanced efficacy of a combinatorial approach involving drug-mediated chemotherapy and rAAV vector-mediated cancer gene therapy to target human hepatocellular carcinoma. In these studies, we identified that shikonin, a naphthoquinone, inhibits human liver tumor growth as well as significantly enhances the efficacy of rAAV vectors carrying a therapeutic gene, tricosanthin (TCS), in a murine xenograft model in vivo. Although our initial mechanistic studies suggested that shikonin-mediated enhancement of rAAV vector transduction was through its proteasome inhibitory activity, which was similar to that of MG132, a specific proteasome inhibitor. However, no further increase was observed when cells were co-treated with both shikonin and MG132.Since shikonin is a multifunctional compound, we wished to evaluate its effect on rAAV vector-mediated transgene expression in additional human cell types. In a human erythroleukemia cell line, K562, frequently used as a model for hematopoietic cell transduction studies, shikonin enhanced the transduction efficiency of various rAAV serotype vectors by up to 10-fold. Surprisingly, however, MG132 had no effect in K562 cells, even at the highest concentration that did not induce cytotoxicity. Thus, in a systematic study, various proteasome and protease inhibitors were evaluated. Western blot assays for the detection of polyubiquitinated total cellular proteins were performed to confirm the effectiveness of these drugs in K562 cells. Whereas carfilzomib only had a modest effect (

Recombinant adeno-associated virus (AAV) vectors are currently in use in a number of Phase I/II clinical trials. However, large vector doses are needed to achieve therapeutic effects. The ubiquitinproteasome pathway plays a critical role in the intracellular trafficking of AAV vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. We have previously reported that substitution of critical amino acid residues dramatically increases the transduction efficiency of vectors based on AAV serotypes 2, 3, 5, and 8 by preventing capsid phosphorylation and subsequent proteasomal degradation.In the present studies, we document that the combination of mutations of specific tyrosine (Y), serine (S), threonine (T) and lysine (K) residues on the capsid of AAV6 vectors also significantly increases the transduction efficiency compared with the wild-type (WT) AAV6 vectors. AAV6 capsid contains 7 tyrosine, 17 serine, 19 threonine and 11 lysine residues, which are surface-exposed. Initially, 17 single-mutants were generated based on the AAV6 crystal structure and overlap of amino acid positions with previously studied serotypes: Y445F, Y705F, Y731F, S455V, S472V, S499V, S551V, S663V, S669V, T251V, T492V, T665V, T722V, K531E, K567E, K666E, K707E. Next, the best performing Y-, S-, T-, and K- mutations were combined onto a single AAV6 capsid to generate 9 double-mutants: Y705+731F, Y731F+T492V, T492V+S663V, T492V+S551V, T665V+S551V, S663V+S551V, T492V+K531E, S663V+K531E, T492V+K666E; 7 triple-mutants: Y445+705+731F, Y705+731F+S663V, Y705+731F+S551V, Y705+731F+T492V, Y705+731F+K531E, Y705+731F+K666E, T492V+S663V+K531E; and two quadruple-mutants: Y705+731F+T492V+S663V, and Y705+731F+T492V+K531E. We identified several AAV6-mutant vectors, such as S663+551V, Y705+731F+T492V and Y705+731F+K531E, which were particularly efficient. These vectors increased the transduction efficiency by approximately 5- to 8-fold in a murine hepatocyte cell line, H2.35, and approximately 3- to 5-fold in a murine myoblast cell line, C2C12, over the WT AAV6 vector in vitro. Similar results were obtained in murine hepatocytes and muscle in vivo after tail-vein or intra-muscular injections of these vectors, followed by whole-body bioluminescence imaging of C57BL/6 mice.These residues are localized towards the base of the protrusions surrounding the icosahedral 3-fold axes of the capsid (T492S, S551V and K531E), in the depression surrounding the 2-fold axes (Y705F and Y731Y), or the HI loop (Y663S). This study thus identifies these capsid surface regions as being critical for the trafficking and transduction properties of the AAV6 vectors.In summary, our studies have led to the generation of novel capsid-optimized AAV6 vectors for their potential use in liver-directed, and muscle-directed gene therapy in humans.

Cancer is a leading cause of human death worldwide. Commonly used methods of treatment such as surgery, radiation and chemotherapy tend to be largely successful but are extremely invasive and have serious toxic side effects. Thus, oncolytic viruses represent an attractive alternative to currently used anti-cancer treatments. Among different viral methods for gene delivery, vectors based on a human parvovirus, the adeno-associated virus (AAV) has attracted much attention mainly because of its non-pathogenic nature, and its ability to mediate long-term, sustained therapeutic gene expression.In current studies, AAV serotype 6 was identified to be particularly effective in transduction of several human prostate (PC3), breast (T47D) and liver (Huh7) cancer cell. We also found that transduction efficiency of these vectors can be significantly improved, up to 5-fold, by site-direct mutagenesis of surface exposed residues involved in intracellular trafficking. Over thirty single and multiple tyrosine (Y), serine (S), threonine (T) capsid mutants were developed and AAV6-Y705-731F+T492V was identified the most efficient.However, promiscuity of the AAV vectors makes it difficult to use in vivo. The recent studies on AAV crystal structure have identified specific regions of the capsid which are surface-exposed and tolerant to insertion of the peptides. In the current studies, we developed novel AAV-based vectors for targeting of human cancer cells by incorporating Arg-Gly-Asp (RGD) containing peptide to enable AAV to infect integrin-expressing cells. These AAV6-RGD vectors improve transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing viral entry to the cells. We correlated increased transduction efficiency with levels of expression of avβ5 integrin.The combination of RGD-peptide and mutations of surface-exposed residues on a single AAV6 capsid, further increases transduction efficiency up to approximately 10-fold. We next used qPCR analysis to quantitate the vector genomes in cytoplasmic and nuclear fractions of cells infected with the WT and the mutant AAV6 vectors at different time points. We showed that the increase in the transduction efficiency of the AAV6-Y705-731F+T492V vector correlated well with the improvement of both the entry and nuclear translocation of the vectors, compared with AAV6-WT.To conduct animal studies, we also introduced an additional mutation at lysine (K531E) since this was shown to impair the ability of vectors to bind to heparan sulfate proteoglycan and thus decrease the affinity to mice hepatocytes. We used a xenograft animal model in immune-deficient mice to prove the specificity and high transduction efficiency of AAV6-RGD-Y705-731F+T492V-K531E vectors in vivo.In summary, this approach can potentially lead to the development of therapeutic AAV vectors with selective tropism and high transduction efficiency of human cancer cells.

Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al., 2002, Hum. Gene Ther. 13, 1935–1943) found limited applications due to the system’s design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passage-dependent loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability and to extend the baculovirus platform to other rAAV serotypes, we have modified both Rep- and Cap-encoding components of the original system. The modifications include a parvoviral phospholipase A2 domain swap allowing production of infectious rAAV8 vectors in vivo. Alternatively, an infectious rAAV8 (or rAAV5) vector incorporating the AAV2 VP1 capsid protein in a mosaic vector particle with AAV8 capsid proteins was produced using a novel baculovirus vector. In this vector, the level of AAV2 VP1 expression is controlled with a “riboswitch,” a self-cleaving ribozyme controlled by toyocamycin in the “ON” mode. The redesigned baculovirus system improves our capacity for rAAV manufacturing by making this production platform more applicable to other existing serotypes.

We first reported that of the 10 most commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). More recently, two independent groups also reported successful transduction of primary human CD34+ cells using the wild-type (WT) AAV6 vectors (Sci. Transl. Med., 7: 307ra156, 2015; Nat. Biotechnol., 33: 1256-1263, 2015), except that multiplicities of infection (MOIs) ranging from 100,000-200,000 vgs/cell were used to achieve ~40-55% transduction efficiency. Furthermore, the transduction efficiency of the WT AAV6 vector varies greatly in HSPCs from different donors, ranging between ~6-87%. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705F+Y731F+T491V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, ‘cross-transduction’. The combined use of these strategies resulted in transduction efficiency exceeding 90% at an MOI of 20,000 vgs/cell in primary human cord blood-derived HSPCs at day 4 (Fig. 1AFig. 1A). scAAV6 vectors were more efficient than ssAAV6 vectors, but at high cell density, there was a modest enhancement in EGFP-positivity even with ssAAV6 vectors. However, 14 days post-transduction, virtually no EGFP-positive cells could be detected (Fig. 1BFig. 1B), suggesting the loss of vector genomes, and hence, the lack of stable integration of vector genomes in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. *These authors contributed equally to this work #Co-corresponding authorsView Large Image | Download PowerPoint Slide

There is little doubt that AAV did not evolve for the purposes of delivery of therapeutic genes. The use of first generation AAV vectors, albeit successful, is unlikely to reach its full potential. We have described the development of capsid-modified next generation [NextGen] AAV vectors for both AAV2 (Proc Natl Acad Sci USA, 105: 7827-7832, 2008; Mol Ther., 18: 2048-2056, 2010; Vaccine, 30: 3908-3917, 2012; PLoS One, 8: e59142, 2013) and AAV3 (Gene Ther., 19: 375-384, 2012; Hum Gene Ther., 25: 1023-1034, 2014) 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 Fig. 1AFig. 1A). We have also described the development of genome-modified generation X [GenX] AAV vectors (J Virol., 89: 952-961, 2015), in which the transcriptionally-inactive, single-stranded AAV genome was modified to achieve improved transgene expression (Fig. 1BFig. 1B). Thus, we reasoned that encapsidation of GenX AAV genomes into NextGen AAV capsid might lead to further increased transduction at further reduced vector doses. To this end, the following sets of ssAAV2 as well as ssAAV3 serotype vectors containing the firefly luciferase (Fluc) reporter gene were generated: (i) wild-type (WT) genome and Y444+500+730F+T492V AAV2 quadruple-mutant (QM) capsid (WT-Fluc-AAV2/QM); (ii) two GenX genomes and AAV2 QM capsid (LC1-Fluc-AAV2/QM and LC2-Fluc-AAV2/QM); (iii) WT genome and S663V+T492V AAV3 double-mutant (DM) capsid (WT-Fluc-AAV3/DM); and (iv) two GenX genomes and AAV3 DM capsid (LC1-Fluc-AAV3/DM and LC2-Fluc-AAV3/DM). The combination of the modified-genomes with the capsid-mutants led to ~5-6-fold increase with both AAV2 and AAV3 serotype vectors following transduction of a human hepatocellular carcinoma (HCC) cell line, Huh7, at an MOI of 1,000 vgs/cell under identical conditions in vitro. When male C57BL/6 mice were injected via tail-vein with WT-Fluc-AAV2/QM, LC1-Fluc-AAV2/QM, and LC2-Fluc-AAV2/QM vectors at a relatively low dose of 5×109 vgs/mouse, the AAV2 QM capsid-mutant vectors led to ~6-10-fold increase in transgene expression in the liver. Similarly, when WT-Fluc-AAV3/DM, LC1-Fluc-AAV3/DM, and LC2-Fluc-AAV3/DM vectors were injected via tail-vein in NSG mice xenografted with human liver tumors at relatively low dose of 3×109 vgs/mouse, led to ~5-8-fold increase with the combination of modified-genomes with the AAV3 DM capsid-mutant vectors, which was restricted to human liver tumors. Taken together, these data document that the combination of NextGen capsids and GenX genomes leads to the generation of optimized [Opt] AAV serotype vectors (Fig. 1CFig. 1C), which transduce cells and tissues more efficiently, both in vitro and in vivo, at significantly reduced doses. These studies have significant implications in the potential use of the Opt AAV serotype vectors in human gene therapy.View Large Image | Download PowerPoint Slide

Cancer, a leading cause of death in the human population today, is generally treated with rigorous surgery followed by intensive chemotherapeutic methods, both of which have adverse toxic side effects. Immunotherapy represents an attractive alternative since it is based on activation of the host immune system. We recently showed that capsid-optimized AAV6-S663V+T492V vectors expressing a tumor-associated antigen are able to initiate specific peripheral T-cell response and suppress tumor progression as well as extend survival in a prostate cancer animal model (J Immunother., 38: 292-298, 2015). However, vaccination based on AAV6 vectors alone was not able to completely overcome the adverse effect of tumor microenvironment and tumor-induced immune dysfunction. Tumor-associated macrophages (TAMs) are prominent components of solid tumors. TAMs exhibit distinct phenotypes and function, and negatively contribute to immune system evasion. Previously published data indicate that inhibition of lipoxygenase (15-LOX2), a key enzyme contributing to TAM function, is critical to overcome tumor resistance and facilitate immunotherapy treatment. In our studies presented here, we first showed that 15-LOX2 is expressed in mouse prostate cancer cells, RM1, growing subcutaneously in C57BL/6 mice, but not in cells growing in culture. We also showed that LOX inhibitors, such as a plant-derived compound, nordihydroguaiaretic acid (NDGA), could restore the function of specific cytotoxic T-cells co-cultured with TAMs in a concentration-dependent manner. Splenocytes from C57BL/6 mice injected intra-muscularly with capsid-optimized AAV6 vectors expressing prostatic aid phosphatase (PAP) were isolated 14 days after vector injection. T-cells were incubated in the presence or absence of RM1 tumor cell extracts and NDGA for 2 hours and then used in killing assays against cultured RM1 cells. Two-color fluorescence assays of cell-mediated cytotoxicity was used to estimate the percentage of dead/alive target cells and a killing curve was generated with different effectors to target cell ratios. Our data indicated that the killing ability of T-cells initiated by capsid-optimized AAV6 vector injection and incubated with RM1 tumor-derived TAMs was approximately 3-fold lower than original activity T-cell. NDGA at a concentration of 10 uM had limited effect on the T-cell activity, but 25 µM NDGA restored the function of the T-cells to approximately 90% of the original activity. In our currently ongoing studies, we are using a combination of capsid-optimized AAV6 vector-mediated immune activation with the NDGA-treatment to corroborate whether a synergetic therapeutic effect can be achieved in an animal model. If successful, this approach could prove useful in the potential clinical application.

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.

Current challenges for recombinant adeno-associated virus (rAAV) vector–based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer.

The human embryonic kidney cell line, HEK293, is readily transduced by the most commonly used AAV2 serotype vectors because these adherent cells abundantly express the cell surface receptor, heparan sulfate proteoglycan (HSPG), and one of the co-receptors, human fibroblast growth factor receptor 1 (FGFR1) for AAV2. However, a human erythroleukemia cell line, K562, commonly used as a model for human hematopoietic cell transduction, is not transduced as efficiently, although these cells grown in suspension, express both HSPG and FGFR1 only at modestly lower levels. We reasoned that the lack of proximity of HSPG and FGFR1 on K562 cells might account for the suboptimal transduction of these cells, and hypothesized that if the transduction was performed at high cell density, presumably allowing for HSPG on one cell to come in close proximity to FGFR1 on the neighboring cell, then AAV2 bound to HSPG on one cell could utilize FGFR1 on the neighboring cell to gain entry in the latter, and vice versa, thus leading to increased transduction. To test this hypothesis, HEK293 and K562 cells were transduced with 3×103 vgs/cell of scAAV2-EGFP vectors either at low-density (60,000 cells in 50 ml) or at high-density (480,000 cells in 50 ml). Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. Whereas ~90% of HEK293 cells expressed the transgene at low-density, the transduction efficiency at high-density increased up to ~95%. On the other hand, only ~35% of K562 cells were transduced at low-density, but interestingly, the transduction efficiency at high-density increased to ~65%, thus corroborating that close cell-cell contact facilitated vector entry into these cells. That the initial cell-cell contact was critical in achieving improved transduction, was further corroborated by experiments in which cells were transduced at low-density, and subsequently pooled together to reach high-density, and conversely, cells were transduced at high-density, and soon after transduction, were diluted to low-density. The increased transduction was observed only under the latter condition. Similar results were obtained with two additional human cell lines, M07e and Raji, which express low to extremely low levels of HSPG and FGFR1, respectively, and consequently, are transduced extremely poorly by AAV2 vectors. These studies were also extended to include primary human CD34+ hematopoietic stem/progenitor cells (HSPCs), also grown in suspension, and known to be transduced sub-optimally by AAV2 vectors. In these studies also, whereas only~5% of these cells were transduced at low-density, the transduction efficiency increased up to ~20% at high-density. Taken together, our studies have revealed a novel mechanism, which we have termed “cross-transduction”, which AAV vectors exploit to gain entry into target cells, and which may have implications in the optimal use of AAV vectors in human gene therapy applications.

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). However, the transduction efficiency of these vectors ranged between ~6-87% in HSPCs obtained from several different donors (n=11). Such a wide range of transduction efficiency of AAV6 vectors is presumably due to different levels of expression of the putative receptors and/or co-receptors on these cells. In our more recent studies, we observed that the transduction efficiency of AAV2 vectors could be augmented both by performing transduction of HSPCs with the wild-type (wt)-AAV2 vectors at high cell density, or by using capsid-modified Y444F+Y500F+Y731F+T491V-mutant AAV2 vectors. In the present studies, we examined whether similar strategies could also be employed to increase the transduction efficiency of HSPCs from donors that are not transduced efficiently by wt-AAV6 vectors. Primary human HSPCs were transduced with either 3×103 or 3×104 vgs/cell of scAAV6-EGFP vectors either at low-density (20,000 cells in 50 ml) or at high-density (200,000 cells in 50 ml). scAAV2-EGFP vectors were also used for comparison. Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results showed that whereas only ~14% of the cells transduced at low-density with high moi expressed the transgene, the transduction efficiency at high-density increased up to ~20% and 25%, at low, and high mois, respectively, also with a significant increase in the mean fluorescence intensity, thus corroborating that the initial cell-cell contact was a critical factor in achieving increased transduction. Next, the transduction efficiencies of the wt- and the capsid-modified triple-mutant (Y705F+Y731F+T492V) AAV6 vectors were compared. Again, the wt- and the capsid-modified quadruple-mutant (y444F+Y500f+Y731F+T491V) AAV2 vectors were used for comparison. HSPCs were transduced with 1×104 vgs/cell at high-density (200,000 cells in 50 ml), and transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results also clearly documented that the ~27% transduction efficiency of the wt-AAV6 vectors was increased by up to ~45% with the capsid-modified AAV6 vectors, with a concomitant increase in the mean fluorescence intensity. Taken together, these studies further corroborate the novel mechanism of “cross-transduction” by recombinant AAV vectors of human cells in general, and HSPCs in particular. Additional human HSPC transplant studies in a murine xenograft model in vivo are currently underway, the successful completion of which is likely to have significant implications of these strategies in the optimal use of AAV6 vectors in the potential gene therapy of human disorders involving the hematopoietic system.

Objective In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro , and in murine hepatocytes in vivo . Methods Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. Results We observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro , and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. Conclusion These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

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.

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34(+) cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2-3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.

Two additional grants from the National Eye Institute were incorrectly omitted from the Funding Statement. The numbers of the two grants are: R24EY022012 and R01EY017549.