Liver Transduction Research Articles

Abstract 3534: Development of the novel AAV-based vectors with selective tropism to human cancer cells

Abstract 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. The possibility of manipulating viral genomes and the natural ability of viruses to efficiently infect various cell types has increased an interest in using a virus-based delivery system to express cytotoxic products within target cells. 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 identified AAV serotype 6 being particularly effective in transduction of several human prostate (PC3), breast (T47D) and liver (Huh7) cancer cell in vitro. We also found that transduction efficiency of these vectors can be significantly, up to 3-5-fold, improved by site-direct mutagenesis of surface exposed residues involved in intracellular trafficking. Over thirty single tyrosine (Y), serine (S), threonine (T) and multiple capsid mutants were developed and AAV6-Y705-731F+T492V was identified the most efficient. However, the vector promiscuity makes it difficult to use in vivo. In the more recent studies on AAV crystal structure, combined with various site-directed and insertion mutagenesis studies of the capsid gene, have identified specific regions of the capsid proteins that 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 for approximately 3-fold compare with wild-type AAV6 vectors by enhancing viral entry to the cells. Combination of RGD-peptide and mutations of surface-exposed residues on a single AAV6 capsid further increases transduction efficiency up to approximately 8-10-fold. We also introduced additional mutation at lysine (K531E) since this was shown to impart a heparan sulfate proteoglycan binding ability of vectors and, thus, decrease affinity to mice hepatocytes. We are currently conducting studies on xenograft animal model in immune-deficient mice to prove specificity and high transduction efficiency of human cancer cells by AAV6-RGD-Y705-731F+T492V-K531E vectors in vivo. In summary, this approach potentially can lead to the development of therapeutic AAV vectors with selective tropism to human cancer cells. Citation Format: Rana Sayroo, Zifei Yin, Diego Nolasco, Munjal Pandya, Chen Ling, George Aslanidi. Development of the novel AAV-based vectors with selective tropism to human cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3534. doi:10.1158/1538-7445.AM2015-3534

Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

311. AAV8 Vexosome Vectors Enhance Cell Transduction In Vitro and Outperform Conventional AAV8 Vectors in Liver-Transduction In Vivo in the Presence of Anti-AAV Neutralizing Antibodies

Adeno-associated virus vectors based on serotype 8 (AAV8) have demonstrated superior efficiency of transduction of hepatocytes in vivo in animal models and in humans. Recently, we described extracellular vesicle (exosomes) -associated AAV vectors termed vexosomes (vAAV). We tested vAAV1, 2, and 9 and found them to enhance transduction and antibody evasion capabilities compared to conventional AAV vectors.With the goal of developing an enhanced vector for liver gene therapy, we sought to characterize the in vitro and in vivo transduction, biodistribution, and immunogenicity profile of vAAV8 vectors compared to conventional AAV8 vectors purified from the same preparation.Several cell lines were transduced with conventional AAV8 or vAAV8 vectors expressing luciferase, at multiplicity of infection (MOI) ranging from 250 to 25000, and luciferase expression was measured after an overnight incubation. In HeLa and HEK293 cells, levels of luciferase up to 1 log higher were observed with vAAV8 compared with AAV8 vectors, with detectable levels of luciferase at MOIs as low as 250. In hepatocyte cell lines, the magnitude of enhancement was about 3-fold, with lower max relative light unit signal measured compared to the other cell lines. Notably, although at low levels, transduction with vAAV8 vectors was also observed in a Jurkat T cell line, in which no transduction was observed with conventional AAV8 vectors.We then evaluated the efficiency of liver targeting in vivo with this vector system using the secreted factor IX (F.IX) transgene. Conventional or vAAV8 vectors expressing human F.IX under the control of a liver-specific promoter were administered i.v. to C57BL/6J male mice at 5×1010 vg/mouse. In naive animals, no difference in F.IX transgene expression levels was observed between the two vector types; similarly, vector genome were found at similar levels in liver, spleen, muscle, lungs, heart, and kidneys. In these animals, i.v. vector administration also resulted in identical levels of anti-AAV antibody formation. We next evaluated the effect of vAAV8 on liver transduction in the presence of antibodies. Animals were passively immunized with IVIg intraperitoneally (0.5, 5.0, and 15 mg/mouse) followed by administration of vector (5×1010 vg) intravenously 24 hours later. At a neutralizing antibody titer of~1:10 (0.5mg IVIg), vAAV8 vector-injected mice had levels of expression identical to those in naive animals. At higher titers (>1:100, 5.0 mg), low levels of residual expression of F.IX were measure with vAAV8 vectors and no expression was measured with conventional AAV. No expression was detected with either AAV8 or vAAV8 vectors in animals immunized with the highest dose of IVIg.In conclusion, vAAV8 vectors have unique transduction characteristics that may be exploited in vitro to transduce various cell types. In vivo, these vectors showed identical levels of liver transduction in naive male mice and provided some level of protection from antibody-mediated vector neutralization.

105. A Single Administration of Low Dosage (5 x1010 vp/kg) hFX/HDAd Complex Achieves Saturated hFVIII Levels in the Blood of Mice

The development of helper-dependent adenoviral vectors (HDAds) devoid of all functional viral genes has largely overcome cellmediated host immune responses associated with Ad viral proteins. However, a dose-related acute toxicity associated with systemic delivery has been a major obstacle to the clinical translation of Adbased vectors. Since HDAd has a large transgene cargo (up to 34 kb), we recently succeeded in producing an HDAd encoding two codon-optimized hFVIII variant N6 expression cassettes (HDAd2xcoN6). We evaluated whether HDAd2xcoN6 improves hFVIII expression and activity in vivo. Hemophilia A (HA) mice were injected with a sub-lethal (human) dose (5 x1011 vp/kg) of HDAd2xcoN6 or HDAd1xcoN6 encoding a single expression cassette. HA mice injected with HDAd2xcoN6 exhibited over 10-fold higher hFVIII expression and activity compared to those injected with an equal titer of HDAd1xcoN6. Thus, we evaluated hFVIII expression after a systemic administration of 5 x1010 vp/kg (one log-lower than a human sub-lethal dose) of HDAd2xcoN6 in non-human primates. One primate showed 2% hFVIII levels in plasma compared to pretreatment. Though that expression was transient, it represents the first time that an Ad-based vector has shown a detectable level of therapeutic gene expression in the blood after systemic administration with such a low dosage in a large animal model. However, this result also suggests that the improvement of transduction of HDAd and transgene expression in the liver is required for long-lasting, high hFVIII expression in a large animal model.Previous reports have revealed that the adenoviral capsid interacts with coagulation factor X (FX) in the blood after systemic administration, and the interaction leads to transduction of this complex into the liver in conjunction with highly expressing HSPGs (receptor of FX/Ad5 complex). The complex of Ad5 with human FX (hFX/HDAd) has a greater affinity to HSPGs than mouse FX does. We therefore evaluated forming a complex of HDAd with hFX in order to improve HDAd liver transduction. Mice injected with 5 x1010 vp/kg of hFX/ HDAd2xcoN6 complex sustained 100% hFVIII levels in the blood until 4 weeks after injection. In contrast, mice injected with same dosage of HDAd2xcoN6 alone had undetectable levels of hFVIII in the blood 4 weeks post-injection, similar to our observations in non-human primate. Although mice injected with the hFX/HDAd complex showed 10-fold higher transgene products in vivo compared to mice treated with HDAd alone, HDAd vector copies in the livers of mice injected with the hFX/HDAd complex were only 2-fold higher. Interestingly, mice injected with the hFX/HDAd complex exhibited 10-fold higher transgene expression at mRNA level in the liver compared to mice injected with HDAd alone. These results suggest that the hFX/HDAd complex can lead not only to high liver transduction, but also to improving transgene expression at the transcriptional level after systemic administration. We are now investigating how the hFX/HDAd complex improves transgene expression at the transcriptional level.

83. Adenoviral Mediated Compartmentalized Liver Transduction Improves Safety and Yields Long-term Transgene Expression in a Porcine Model

To stringently control the viremia associated with conventional routes of vector administration and significantly reduce the astronomical quantities of virus commonly infused in gene therapy, our group described compartmentalized liver transduction. Compartmentalized liver transduction is based on known adenoviral spatio-temporal kinetics and is achieved by the intra-parenchymal injection of a vector dose into a blood flow isolated portion of the liver; perfusion is reestablished after viral endocytosis has been completed (30 min). Given the encouraging results documented in rodents (rats), our group assessed compartmentalized liver transduction in a porcine model. To this end, a surgical device was conceptualized, prototyped and utilized to safely and securely transiently isolate a portion of the liver in 25- 30 kg Vietnamese pigs. Implementing compartmentalized liver transduction in pigs, no viremia was detected during the procedure and transgene expression and presence were confined to the site of vector injection. Neither inflammation, apoptosis or significant biochemical liver damage were observed. Using porcine alpha fetoprotein as a reporter gene, serum levels were documented with low quantities of vector dose (10^5) and prolonged transgene expression was achieved (10 months). Our results further support that compartmetalized liver transduction has profound paradoxic effects on the classic toxicological interactions between the liver and adenovirus. The lack of acute liver inflammation observed suggests that conventional immunolgical thresholds and relays were not triggered. We further postulate that compartmentalized liver transduction induces the local synthesis of tolerance inducing cytokines central to thwarting the acute, intermediate and late adenoviral induced immune responses and are critical in generating an immune priviledge liver compartment capable of long term protein synthesis and secretion. Our results further demonstrate that compartmentalized liver transduction is a safe and effective route of vector delivery with a promising future in the field of gene therapy.

323. Adenoviral Compartmentalized Liver Transduction Improves Safety and Yields Longerm Transgene Expression

Still unresolved complications associated with adenoviral mediated hepatic-based gene therapy are a strong immunological response with damage to the organ and short-term gene expression. Our group hypothesized that the two components at the core of these drawbacks are: 1) the astronomically high quantities of vector infused and 2) the viremia associated with the conventional routes of vector administration. To stringently manipulate these two variables and assess their effect on adenoviral mediated liver transduction our group implemented compartmentalized liver transduction. Compartmentalized liver transduction is based on known adenoviral spatio-temporal kinetics and is achieved by the direct intra-parenchymal injection of adenovirus into a blood-flow isolated portion of the liver; blood flow is reestablished after viral endocytosis has been completed. Implementing compartmentalized liver transduction in a rat model, gene expression and transgene presence was confined to the site of injection, no inflammation nor liver damage was observed, reporter protein expression with low quantities of vector (10^2) as well as linear dose response curves were documented and prolonged transgene expression was achieved. Our results conclude that compartmentalized liver transduction is a safe and effective mode of vector delivery with a promising future in the field of gene therapy.

313. Development of the AAV6-Based Vectors With Selective Tropism and High Transduction Efficiency of Human Cancer Cells

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.

264. Absence of CD8 T Cell Responses Against AAV Capsid Using Reduced Vector Dose or Library Selected Liver Targeted AAV2 Based Capsids

Predicting immune responses directed against the AAV capsid have proven to be a challenge in the translation of liver directed AAV gene therapy into the clinic. Previously we have described the first mouse model that allows us to evaluate the potential for CD8 T cell responses directed against AAV transduced hepatocytes and to define protocols and conditions to minimize this response (Martino et al. Blood 2013). Empirical data from both AAV2 and AAV8 clinical trials for hemophilia B have suggested that a threshold vector dose is needed for activation of capsid specific CD8 T cells and subsequent elimination of factor IX expressing hepatocytes. We sought to experimentally confirm this observation in our murine model by evaluating the impact of reducing AAV2 and AAV8 vector doses by one log. No reduction in FIX expression was observed for either AAV2 or AAV8 transduced mice receiving 1×1010 vg and capsid specific CD8 T cells, suggesting that that the reduced vector dose dropped the number of viral particles below a threshold for efficient MHC I presentation.In another set of studies we evaluated two recently described AAV2 capsid variants (AAV2-LiA and AAV2-LiC), which were selected for murine hepatocyte tropism from a highly complex rationally designed capsid library (Marsic et al. Mol Ther 2014). Both AAV2-LiA and AAV2-LiC vectors were not targeted by capsid specific CD8 T cells and resulted in stable FIX expression for the duration of the experiment. Our previous studies conducted on the AAV2-M3 (Y-F 444,500,730) capsid showed that these three mutations provided sustained FIX expression in the presence of capsid CD8 T cells. By design, AAV2-LiA contains two of the previously described (Y-F) mutations at positions 444 and 500 and AAV2-LiC contains one at position 500. These data now extend on this study and suggest that either a single Y-F mutation at position 500 or double at positions 444 and 500 may be sufficient in reducing capsid antigen presentation on hepatocytes. Although it is unclear if these single and double Y-F mutants are alone sufficient or if there are synergistic mutations in AAV2-LiA and AAV2-LiC that contribute to reduced capsid antigen presentation. Nonetheless, we were unable to detect the presence of infiltrating CD8 T cells both at the one week and four week time points where previous studies conducted on a similar AAV2 vector demonstrated loss of FIX expression and the presence of infiltrating CD8 T cells at the one week time point. Interestingly although AAV2-LiA was less efficient than AAV2-LiC in transgene expression in immune competent mice, our studies conducted in immune deficient mice showed that both vectors provided similar levels of FIX expression. These data suggest that AAV liver transduction efficiency is altered in immune deficient mice. Therefore, we would offer caution in the analysis and interpretation of AAV transgene expression data generated solely in immune deficient animals.Together, these data support a vector dose dependency threshold for capsid antigen presentation on hepatocyte MHC I molecules and demonstrates that the novel AAV2-LiA and LiC capsids avoid detection by capsid specific CD8 T cells.

704. Long-Term Correction of Crigler-Najjar Syndrome and Scale-Up Production of an Optimized AAV8 Vector Expressing the UGT1A1 Transgene

Crigler-Najjar syndrome (CN) is an autosomal recessive rare disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene. In severe CN, lack or reduced activity of UGT1A1 results in high levels of serum unconjugated bilirubin (UCB), which can lead to brain damage and death. Treatment of CN consists of phototherapy for 10-12 hours per day to convert UCB into soluble photoisomers without the need of conjugation, which presents several limitations and has a major impact on life quality of the patients. Liver transplantation is the only curative option for CN. The limited therapeutic options available prompted us to develop a new therapy for CN based on the transfer of a corrected copy of the UGT1A1 gene to hepatocytes.We developed an AAV8 vector optimized for the liver expression of the hUGT1A1 transgene (AAV8-hUGT1A1). Safety and efficacy of correction of total serum bilirubin (TB) levels with AAV-hUGT1A1 were demonstrated in mouse and rat models of CN at vector doses as low as 5×10 11 vector genomes (vg)/kg, a result confirmed also by the detection of conjugated bilirubin in bile of treated rats. In juvenile CN rats, long-term correction of total bilirubin levels were observed for more than 8 months following AAV8-hUGT1A1 gene transfer. In neonate Ugt1a1–/– mice, intraperitoneal delivery of the vector at doses as low as 1×10 9 vg/mouse resulted in correction of TB at 4 weeks. However, vector delivery in P2 and P4 animals resulted in lower efficiency of correction when compared with P11-transduced mice, a finding likely due to the more advanced development of the liver at later time points.Based on these data, dose finding studies were performed in CN rats, which showed a profile of liver transduction with AAV8 vectors similar to humans, i.e. lower efficiency of transduction than mice. No differences in efficacy of correction of TB were observed in male vs. female rats.A scalable process for the production of AAV8-hUGT1A1 production was established based on a protocol of triple transfection of suspension HEK293 cells, leading to high yield vector preparations with excellent purity profile. In vitro transduction assays in human hepatocytes based on Western blot for hUGT1A1 protein and in vivo studies in CN rats demonstrated that vectors produced with the suspension process have potency characteristics undistinguishable from research grade vectors produced by triple transfection of adherent cells and purified by cesium chloride gradient centrifugation.In conclusion, our data demonstrate the safety and efficacy of gene transfer for Crigler-Najjar syndrome in two relevant animal models of the disease and provide tools and a strong rationale for the translation of these results in human subjects.

Manipulating adenovirus hexon hypervariable loops dictates immune neutralisation and coagulation factor X-dependent cell interaction in vitro and in vivo.

Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.

Impact of natural IgM concentration on gene therapy with adenovirus type 5 vectors.

Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.

SR-A and SREC-I binding peptides increase HDAd-mediated liver transduction.

Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes without inducing chronic toxicity. However, vector therapeutic index is narrow because of a toxic acute response with potentially lethal consequences elicited by high vector doses. Kupffer cells and liver sinusoidal endothelial cells (LSECs) are major barriers to efficient hepatocyte transduction. We investigated two small peptides (PP1 and PP2) developed by phage display to block scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I) respectively, for enhancement of HDAd-mediated hepatocyte transduction efficiency. Pre-incubation of J774A.1 macrophages with either PP1 or PP2 prior to HDAd infection significantly reduced viral vector uptake. In vivo, fluorochrome-conjugated PP1 and PP2 injected intravenously into mice co-localized with both CD68 and CD31 on Kupffer cells and LSECs, respectively. Compared to saline pre-treated animals, intravenous injections of both peptides prior to the injection of an HDAd resulted in up to 3.7- and 2.9-fold increase of hepatic transgene expression with PP1 and PP2, respectively. In addition to hepatocyte transduction, compared to control saline injected mice, pre-treatment with either peptide resulted in no increased levels of serum interleukin-6 (IL-6), the major marker of adenoviral vector acute toxicity. In summary, we developed small peptides that significantly increase hepatocyte transduction efficacy and improve HDAd therapeutic index with potential for clinical applications.

Harnessing high density lipoproteins to block transforming growth factor beta and to inhibit the growth of liver tumor metastases.

Transforming growth factor β (TGF-β) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFβ-inhibitory molecules. We constructed a plasmid encoding a potent TGF-β-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-β. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-β signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2−/−IL2rγ−/− immunodeficient mice. This effect was associated with downregulation of TGF-β target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8+ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-β-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms.

313. Liver Transduction and Brain Safety of an Antifibrogenic shRNA for Cannabinoid Receptor CB1 in a Model of Liver Cirrhosis

313. Liver Transduction and Brain Safety of an Antifibrogenic shRNA for Cannabinoid Receptor CB1 in a Model of Liver Cirrhosis

240. Efficient and Targeted Transduction of Nonhuman Primate Liver With Optimized AAV3B Vectors Through Systemic Delivery

240. Efficient and Targeted Transduction of Nonhuman Primate Liver With Optimized AAV3B Vectors Through Systemic Delivery

308. Natural Antibodies Inhibit Liver Transduction with Adenovirus Vectors in Mice

308. Natural Antibodies Inhibit Liver Transduction with Adenovirus Vectors in Mice

581. Enhanced Liver Transduction With Novel AAV Resistant To Pre-Existing Immunity in Humans

581. Enhanced Liver Transduction With Novel AAV Resistant To Pre-Existing Immunity in Humans

314. Variations in Liver Transduction Ability Among Species C Adenovirus Vectors

314. Variations in Liver Transduction Ability Among Species C Adenovirus Vectors

Feasibility and Safety of Systemic rAAV9-hNAGLU Delivery for Treating Mucopolysaccharidosis IIIB: Toxicology, Biodistribution, and Immunological Assessments in Primates

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease caused by autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In anticipation of a clinical gene therapy treatment for MPS IIIB in humans, we tested the rAAV9-CMV-hNAGLU vector administration to cynomolgus monkeys (n=8) at 1E13 vg/kg or 2E13 vg/kg via intravenous injection. No adverse events or detectable toxicity occurred over a 6-month period. Gene delivery resulted in persistent global central nervous system and broad somatic transduction, with NAGLU activity detected at 2.9-12-fold above endogenous levels in somatic tissues and 1.3-3-fold above endogenous levels in the brain. Secreted rNAGLU was detected in serum. Low levels of preexisting anti-AAV9 antibodies (Abs) did not diminish vector transduction. Importantly, high-level preexisting anti-AAV9 Abs lead to reduced transduction in liver and other somatic tissues, but had no detectable impact on transgene expression in the brain. Enzyme-linked immunoabsorbent assay showed Ab responses to both AAV9 and rNAGLU in treated animals. Serum anti-hNAGLU Abs, but not anti-AAV9 Abs, correlated with the loss of circulating rNAGLU enzyme. However, serum Abs did not affect tissue rNAGLU activity levels. Weekly or monthly peripheral blood interferon-γ enzyme-linked immunospot assays detected a CD4(+) T-cell (Th-1) response to rNAGLU only at 4 weeks postinjection in one treated subject, without observable correlation to tissue transduction levels. The treatment did not result in detectable CTL responses to either AAV9 or rNAGLU. Our data demonstrate an effective and safe profile for systemic rAAV9-hNAGLU vector delivery in nonhuman primates, supporting its clinical potential in humans.

Evaluation of Prostate-Specific Membrane Antigen as an Imaging Reporter

Genetic reporters provide a noninvasive method to monitor and evaluate a population of cells. The ideal properties of a gene reporter-probe system include biocompatibility, lack of immunogenicity, low background expression or signal, and high sensitivity of detection. The prostate-specific membrane antigen (PSMA) is an attractive candidate for a genetic reporter as it is a human transmembrane protein with a selective expression pattern, and there are several PSMA imaging agents available for clinical and preclinical applications. We evaluated the use of PSMA as a genetic imaging reporter by comparison to 2 clinically established reporters, the mutant herpes simplex virus type I thymidine kinase and the human sodium-iodide symporter. Adenoviruses expressing each reporter were constructed and validated in vitro for expression and function. To compare PSMA with existing imaging reporters, a bilateral Matrigel suspension model was established with nude mice bearing cells equally infected with each reporter or control adenovirus. Dynamic PET was performed, and time-activity curves were generated for each reporter-probe pair. A comparison of peak target-to-background ratios revealed that PSMA offered the highest ratio relative to the control Matrigel suspension as well as muscle. Further, as proof of concept, PSMA was applied as an imaging reporter to monitor adenoviral liver transduction with both nuclear and optical imaging probes. These preliminary studies support further development of PSMA as a noninvasive genetic reporter.