Liver-directed Expression Research Articles

Liver-Directed but Not Muscle-Directed AAV-Antibody Gene Transfer Limits Humoral Immune Responses in Rhesus Monkeys

A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-simian immunodeficiency virus (SIV) monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed, and long-term AAV-mediated delivery has been inconsistent. Here, we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 immunoglobulin G1 (IgG1) delivery using the AAV1 versus the AAV8 serotype with a cytomegalovirus (CMV) promoter and the use of a muscle-specific versus a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer, using a muscle-specific promoter, showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer, using a liver-specific promoter, also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody.

LB.03.05] HELPER-DEPENDENT ADENOVIRUS-MEDIATED GENE TRANSFER OF A SECRETED LDL RECEPTOR/TRANSFERRIN CHIMERIC PROTEIN REDUCES AORTIC ATHEROSCLEROSIS IN LDL RECEPTOR-DEFICIENT MICE

Objective: Familial hypercholesterolemia is a genetic hyperlipidemia characterized by elevated concentration of plasma LDL cholesterol. Statins are not effective in all patients whose prognosis is still quite poor. In the past, we have developed safe and effective gene therapy strategies for the expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We recently developed a HD-Ad vector for the expression of a secreted chimeric protein containing the extracellular portion of the human LDL receptor (LDLR) fused with a transferrin dimer. These project would represent a constitute proof of concept of the possibility of lowering LDL-C and reducing atherosclerosis using a secreted transgene and an alternative intracellular route. Design and method: Development of an HD-Ad vector for liver-directed expression of the TF-LDLR, a fusion protein that mimicks the function of the LDLR; Evaluation of the effect of the TF-LDLR fusion protein in the FH cellular model and mouse model Assessment of efficacy of the PEGylated HD-Ad vector expressing TF-LDLR Evaluation of alternative administration routes for the PEGylated HD-Ad vector expressing TF-LDLR fusion protein. Results: We evaluated the efficacy of vector in CHOldla7 cell line,in which we restored the cell ability to uptake of labeled LDL. We administered intravenously 1X10E13 vp/kg of the HD-Ad vector expressing TF-LDLR in LDLR-deficient mice demonstrating the efficacy of the vector in reducing total and LDL cholesterol levels;in addition, expression of TF-LDLR significantly reduced aortic atherosclerotic lesions in treated LDLR-deficient mice compared to controls. Moreover, we administered in VivoTag 750S-Labeled LDL to study biodistribution in LDLR-deficient mice treated with either HD-Ad TF-LDLR or control vector and we found a strong signal in the heart, liver and intestin after Fluorescence molecular tomography analysis. Conclusions: We demonstrated: In vitro expression and efficacy of the TF-LDLR fusion protein; HD-Ad-mediated in vivo expression of TF-LDLR; Improvement of lipid profile in LDLR-deficient mice treated with TF-LDLR. Reduction of aortic atherosclerosis lesions size in LDLR-deficient mice treated with TF-LDLR. Future steps of our project will involve a thorough safety evaluation of our approach and the possibility of administering the HD-Ad vector in its PEGylated version using alternative routes.

Recognition of Intact Donor Class I by Recipient CD8+ T Cells is Required for Tolerance Induction Following Liver-Directed Transgene Expression

Introduction: AAV-mediated expression of donor MHC I (H-2Kb or H-2Kd) in recipient liver induces donor-specific tolerance in a mouse skin transplant model where Kb or Kd are mismatched between donor and host [1]. Recognition of both intact and processed allogeneic class I could contribute to tolerance induction. Aims and Methods: To determine the relative importance of intact and processed class I in tolerance induction, we generated rAAV-Kb-D227K and Kd-D227K vectors where a point mutation in the class I alpha 3 domain abrogates CD8 binding [2] and direct allorecognition but does not interfere with recognition of processed peptides. Expression of WT or mutant class I on hepatocytes was assessed by FACS and IHC. TCR-transgenic T cells recognizing intact (Des-RAG) or processed (TCR75) allogeneic class I were used as reporter cells in adoptive transfer experiments. Skin bearing the mismatched MHC I was grafted onto uninjected recipients or mice inoculated with rAAV encoding WT or D227K mutant Kb or Kd. In some recipients, Tregs were depleted using anti-FR4 or Diphtheria toxin (DT). Interferon gamma production by responding T cells was determined using ELISpot. Results: WT and D227K class I were expressed on hepatocytes at comparable levels. Adoptive transfer assays confirmed that the D227K mutation abrogated recognition of intact Kb by CD8+ Des-RAG T cells, whilst recognition of processed Kd peptides by TCR75-RAG cells was unimpaired. Uninjected B10.BR mice rejected Kb-mismatched 178.3 skin (MST = 16 d), while grafts onto rAAV-Kb-injected mice survived indefinitely (p < 0.001). rAAV-Kb-D227K only slightly prolonged graft survival (MST = 27d, p < 0.05). These results were recapitulated in the B6.Kd to C57BL/6 model. The modest survival prolongation of skin grafts in mice inoculated with Kb-D227K was abrogated when the recipients were treated with anti-FR4 (clone TH6) to deplete Tregs. Similarly, the survival prolongation of Kd-mismatched B6.Kd grafts onto DEREG recipients treated with rAAV-Kd-D227K (MST = 28d compared with 20d in mice receiving DT alone) was blocked when mice receiving Kd-D227K were also treated with DT (MST 18 days, p = 0.0046). Treg depletion did not shorten survival of skin grafts in mice inoculated with WT MHC class I vectors. Interferon-gamma production by unfractionated or CD8-enriched splenocytes from primed C57BL/6 mice in response to B6.Kd stimulators was over fourfold lower in mice inoculated with WT Kd than in those receiving Kd-D227K (p = 0.038). Conclusions: Recognition of intact MHC class I by alloreactive CD8+ T cells is required for tolerance induction via liver-directed expression of donor MHC class I. Indirect recognition of class I allopeptides and generation of Tregs can produce a modest prolongation of class I-mismatched skin graft survival but does not induce tolerance.FigureFigure

Disruption of CD8-Coreceptor Binding Abrogates Tolerance Induction Via Liver-Directed Expression of Donor MHC Class I - A Role for PD-L1?

Introduction. We previously used an adeno-associated viral (rAAV) vector to express donor MHC (H-2Kb) in recipient livers and induce donor-specific tolerance in a mouse skin transplant model. Kb expression in B10.BR mice led to impaired production of IFN-γ in response to Kb, but no substantial deletion of CD8 T cells, suggesting that tolerance was associated with functional silencing. Methods. To determine the role of direct recognition of class I by CD8-dependent T cells in tolerance induction, we generated rAAV encoding a mutant Kb (rAAV-D227K), where Asp at position 227 of the α3 domain is replaced with Lys, abrogating CD8 binding. Expression of Kb, D227K-Kb and PD-L1 on hepatocytes was assessed by FACS and IHC. Kb-bearing 178.3 skin was grafted onto uninjected recipients and mice injected with rAAV-D227K or rAAV-Kb ± PD-L1 blockade. Results. Uninjected B10.BR mice rejected 178.3 skin (MST=16 d, n=6), while grafts onto rAAV-Kb-injected mice survived long term (MST>250, n=5). Survival of 178.3 grafts onto mice injected with rAAV-D227K was only slightly prolonged (MST=27, n=6), suggesting that CD8 coreceptor engagement is needed for tolerance induction. Kb expression in B10.BR livers resulted in increased PD-L1, but this was not seen following Kb-D227K expression, or with Kb expression in C57BL/6 livers, suggesting that alloreactive CD8 T cell activation results in PD-L1 upregulation. Antibody blockade of PD-L1 plus rAAV-Kb treatment resulted in increased ALT levels (721±73) compared to treatment with rAAV-Kb (33±4) or anti-PD-L1 (35±2) alone. Experiments to determine the effect of PD-L1 blockade on skin graft survival are ongoing. Conclusion. Disruption of CD8 coreceptor engagement abolishes tolerance induction via liver-directed expression of donor MHC class I, indicating that direct recognition by CD8-dependent T cells is essential for the process. PD-L1 upregulation accompanied alloreactive CD8 T cell activation and blockade of PD-L1 resulted in hepatitis, suggesting that this molecule might play a role in the functional silencing of CD8 T cells in this model.

286. An Optimized AAV Vector for Liver-Directed Expression of the UGT1A1 Gene in Crigler-Najjar Syndrome, towards Clinical Development

286. An Optimized AAV Vector for Liver-Directed Expression of the UGT1A1 Gene in Crigler-Najjar Syndrome, towards Clinical Development

An engineered U1 small nuclear RNA rescues splicing‐defective coagulation F7 gene expression in mice

BackgroundThe ability of the spliceosomal small nuclear RNA U1 (U1snRNA) to rescue pre-mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. Coagulation factor deficiencies due to splicing mutations are relatively frequent and could therefore benefit from this strategy. However, the effects of U1snRNAs in vivo remain unknown.ObjectivesTo assess the rescue of the F7 c.859+5G>A splicing mutation (FVII+5A), causing severe human factor VII (hFVII) deficiency, by the modified U1snRNA+5a (U1+5a) in a murine model.MethodsMice expressing the human F7 c.859+5G>A mutant were generated following liver-directed expression by plasmid or recombinant adeno-associated viral (AAV) vector administration. The rescue of the splice-site defective pre-mRNA by U1+5a was monitored in liver and plasma through hFVII-specific assays.ResultsInjection of plasmids encoding the U1+5a rescued plasma hFVII levels, which increased from undetectable to ∼8.5% of those obtained with the wild-type hFVII plasmid control. To assess long-term effects, mice were injected with low and high doses of two AAV vectors encoding the FVII+5A splice site mutant as template to be corrected by U1+5a. This strategy resulted in hFVII plasma levels of 3.9 ± 0.8 or 23.3 ± 5.1 ng mL−1 in a dose-dependent manner, corresponding in patients to circulating FVII levels of ∼1–4.5% of normal. Moreover, in both experimental models, we also detected correctly spliced hFVII transcripts and hFVII-positive cells in liver cells.ConclusionsHere we provide the first in vivo proof-of-principle of the rescue of the expression of a splicing-defective F7 mutant by U1snRNAs, thus highlighting their therapeutic potential in coagulation disorders.

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Optimization of Self-complementary AAV Vectors for Liver-directed Expression Results in Sustained Correction of Hemophilia B at Low Vector Dose

Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4-20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread ( approximately 80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.

Adenovirus-mediated gene transfer and lipoprotein-mediated protein delivery of plasma PAF-AH ameliorates proteinuria in rat model of glomerulosclerosis

Oxidative stress has been proposed to play a crucial role in glomerulosclerosis, although its in vivo demonstration has proved taxing given the difficulty of inducing gene expression in specific renal cells. In this study, we examined whether the liver-directed expression of plasma platelet-activating factor acetylhydrolase (PAF-AH) would affect the glomerular pathophysiology in Imai rats, an animal model for glomerulosclerosis. Adenovirus-mediated liver-directed gene delivery of human PAF-AH resulted in a significant increase in plasma PAF-AH activity, which was detected almost exclusively on HDL. Histological examination of rats overexpressing PAF-AH showed not only the deposition of PAF-AH in mesangial cells, but also a reduction in hydroxynonenal and matrix protein content in the glomeruli. In situ hybridization analysis was negative for human PAF-AH mRNA in the kidney, while injection of HDL abundant in PAF-AH resulted in the deposition of PAF-AH in mesangial cells. Urine protein levels did not increase in rats overexpressing PAF-AH, while those of control rats increased significantly with age. This study provides direct evidence of the in vivo role of an enzyme that degrades lipid peroxides during the progression of glomerulosclerosis. Adenovirus-mediated extrarenal gene expression and lipoprotein-mediated glomeruli-targeted protein delivery promise to be a novel therapeutic approach to glomerulosclerosis.

Adeno-Associated Virus Vectors in Clinical Trials

Adeno-Associated Virus Vectors in Clinical Trials

Factor IX variants improve gene therapy efficacy for hemophilia B

AbstractIntramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Liver-directed gene expression employing synthetic transcriptional control units

To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems. Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. A new bifunctionally-enhanced green fluorescent protein (EGFP)/neo(r) fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis. Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.

580. Inducible Expression of Interleukin-12 for the Treatment of Liver Metastases Using a High-Capacity Adenoviral Vector

Background and aims: The cytokine Interleukin-12 (IL-12) has a potent antitumor effect on a variety of cancer models, but its toxicity precludes the systemic administration in humans. The liver-directed expression of IL-12 using adenoviral vectors is a promising alternative for the treatment of primary and metastatic liver cancer. The development of a high-capacity (“gutless”) adenoviral vector with a mifepristone-inducible system for expression of IL-12 (GL-Ad/RUmIL12) can add further efficacy and safety to this approach. Here we describe the kinetics of IL-12 expression, the toxicity and the antitumor effect of GL-Ad/mIL12 on a newly developed tumor model using different routes of administration.

Targeting of therapeutic gene expression to the liver by using liver-type pyruvate kinase proximal promoter and the SV40 viral enhancer active in multiple cell types

To achieve the liver-directed expression in sufficient amounts of therapeutic genes for successful and safe gene therapy, natural liver-specific promoters can be used to direct the expression of therapeutic genes in the liver, whereas strong viral enhancers were used to obtain sufficient amounts of expressed therapeutic gene products. However, very often use of either the former or the latter does not guarantee both potent and liver-specific therapeutic gene expression. Here we conglomerate them and thus create a potent tissue-specific promoter by characterizing and using the liver-type pyruvate kinase proximal promoter (LPKPP) harboring its TATA box and a HNF-1α binding site. Alone it hardly activated its reporter gene expression in non-hepatocytes or hepatocytes. However, in the presence of the SV40 viral enhancer (SV40VE), which is active in multiple cell types, it was able to potently activate its reporter gene expression specifically in hepatocytes. The tissue-specific activation of the LPKPP by the viral enhancer was attributed to HNF-1α binding to the LPKPP. Taken together, these results support the idea that the constitutively active SV40VE could be used to activate the LPKPP in a tissue-specific manner in the presence of HNF-1α. To our knowledge, this is the first study to utilize HNF-1α and its binding site, in the context of the LPKPP, to generate a basal promoter that is transcriptionally activated potently in a tissue-specific manner by a viral enhancer that is active in multiple cell types.

1252. Analysis of Promoters for Liver-Directed Expression

1252. Analysis of Promoters for Liver-Directed Expression