Transgene Expression In Liver Research Articles

Adeno-associated virus (AAV) 6 provides efficient gene transfer upon intramyocardial injection in vivo

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Innovation Council Eurostars Background Cardiac gene transfer utilizing adeno-associated virus (AAV) is a promising approach for the treatment of various cardiovascular diseases. Intramyocardial injection is an efficient delivery route, and is particularly relevant for ischemic disease or pacemaker and conduction system abnormalities, where local treatment is favoured. However, which AAV variants provide efficient cardiac gene transfer after intramyocardial injection remains to be tested. Aim This study aims to compare several cardiotropic AAV variants in order to identify their relative potency in the setting of direct intramyocardial injection. Methods A green fluorescent protein (GFP)-coding sequence was packaged in the two benchmark AAV capsids, AAV6 and AAV9, and a selection of recently reported AAV9 capsid variants which are named MyoAAVs. These AAV vectors were added to neonatal rat ventricular cardiomyocytes (NRVM) and their transduction efficiency was evaluated by flow cytometry and quantitative PCR. AAV6, AAV9 and MyoAAV4A vectors were then delivered into the left ventricle of FVB mice via intramyocardial injection. AAV6 and AAV9 carrying GFP in a different expression cassette were also intramyocardially injected in FVB and Bl6 mice. Direct fluorescence, transgene mRNA, and viral genomes were quantified to determine the transduction efficiency 4 weeks post injection. Picrosirius red staining was used to assess whether AAV-mediated GFP delivery causes cardiac fibrosis. We further studied GFP encoding AAV6 and AAV9 vectors upon intramyocardial injection into the left ventricle of pigs and determined the transduction efficiency by quantitative PCR. Results NRVM transduction experiment revealed that among the tested capsid variants, AAV6 is the most efficient in transducing cardiomyocytes, followed by MyoAAV4A. In mice, AAV6 is most effective in transducing myocardium upon intramyocardial injection, resulting in approximately 10-fold higher transgene expression than that obtained with AAV9 vector and 2-fold higher than that with MyoAAV4A vector. Histology revealed no vector-related cardiac fibrosis. The use of different expression cassettes and animal strains consistently showed that AAV6 outperforms AAV9 in cardiac transduction efficiency. Transgene expression in liver was only observed in Bl6 mice injected with AAV9. In porcine myocardium, AAV6 led to robust transgene expression after intramyocardial injection and this expression level is higher than that of AAV9. Conclusion Among the AAV variants tested, AAV6 appears to be the most efficient for cardiac transduction by intramyocardial injection. These results suggest that AAV6 represents an attractive vector for direct cardiac gene transfer and for future capsid engineering in the context of local transgene delivery.

Therapeutic homology-independent targeted integration in retina and liver

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.

Characterization of integration frequency and insertion sites of adenovirus DNA into mouse liver genomic DNA following intravenous injection.

While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per μg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 1011 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.

Platelet FV Deficiency Restores Survival of TFPI Null Mice Independently of Plasma FV

Introduction: Platelets are essential for the hemostatic process, releasing “just in time” prothrombotic molecules, such as factor V (FV) and prothrombin to sites of vascular injury. Paradoxically, platelets also carry potent antithrombotic molecules, including tissue factor pathway inhibitor (TFPI), which may prevent hemostatic platelet plugs from expanding into vascular occluding thrombi. The TFPI gene undergoes alternative splicing to produce several isoforms, including TFPIα and TFPIβ. Platelets are the primary location of TFPIα in the murine circulation. TFPIα has a basic 9 amino acid sequence in its C-terminal region nearly identical to a basic sequence in the FV B domain. The TFPIα basic region tightly binds to an acidic region of the FV B domain. This binding takes place only when the FV basic region is absent, as occurs following proteolysis by factor Xa (FXa), in some forms of FV naturally present in platelet α-granules, or in genetically altered forms of FV, such as FV-short (associated with East Texas bleeding disorder). This interaction provides an essential exosite for TFPIα inhibition of the prothrombinase complex (FXa-FVa) assembled with forms of FVa retaining the B domain acidic region. Since prothrombinase assembles on the surface of activated platelets, we hypothesized that inhibition of prothrombinase via the platelet TFPIα-platelet FV interaction is a physiologically important biochemical mechanism for the inhibiting early stages of coagulation. We probed this hypothesis using TFPI K1 deleted (TFPI-/-), FV deleted (F5-/-) and FV transgenic (F5Tg) mice to uncover whether specifically reducing or removing platelet FV would rescue TFPI-/- mice from embryonic lethality.Methods/Results: We performed crossbreeding experiments using F5-/- mice carrying liver- or platelet-specific transgenes. F5LiverTg produces 50% plasma FV with no FV in platelets, whereas F5PltTg produces 3% platelet FV with no FV in plasma. F5-/- F5LiverTgand F5-/- F5PltTgmice were bred into TFPI+/- mice. Offspring from this cross were bred to determine the effects of the liver or platelet transgene on the survival of mice lacking TFPI.F5-/- is embryonic lethal in mice. F5-/- mice are rescued by transgenes expressing either liver-specific FV or platelet specific FV. When we bred F5+/- TFPI+/- x F5+/- F5LiverTg TFPI+/-, neither F5-/- TFPI-/- nor F5+/- TFPI-/- mice without the liver transgene were viable. Liver transgene expression did not rescue F5+/- TFPI-/- mice, however, it produced grossly normal pups lacking TFPI (F5-/- F5LiverTg TFPI-/-) that lived to adulthood with no overt phenotypes. We further characterized this phenotype by crossbreeding F5+/- TFPI+/- x F5-/- F5LiverTg TFPI-/-. In this cross, F5-/- F5LiverTg TFPI-/- pups survived with ~80% penetrance (45 of 234 total mice, p<5.0 x10-12). Next, we bred F5+/- TFPI+/- x F5+/- F5PltTgTFPI+/-. Platelet transgene expression also produced grossly normal pups lacking TFPI (F5-/- F5PltTg TFPI-/-) that lived to adulthood with no overt phenotypes. Further crossbreeding, as described for the liver transgene, revealed a similar ~80% survival of F5-/- F5PltTg TFPI-/- pups (56 of 291 total mice, p<9.0 x10-18). Both male and female platelet and liver specific F5-/- F5Tg TFPI-/- were fertile, and mothers supported pups until weaning.Conclusions: Neither total loss of FV nor concerted 50% reduction in plasma and platelet FV rescued TFPI-/- embryonic lethality, indicating that TFPI anticoagulant activity is not simply counterbalanced by FV procoagulant activity (or vice versa) during embryogenesis. Remarkably, either the total lack of platelet FV with 50% normal plasma FV, or the total lack of plasma FV with 3% normal platelet FV, resulted in the nearly complete rescue of F5-/- TFPI-/- mice with pups surviving to adulthood without grossly observable pathology. These observations indicate that severe reduction of platelet FV, independent of the plasma FV concentration, produces this extraordinary rescue of TFPI-/- embryonic lethality. Collectively, these findings expose the critical role for platelet TFPIα in regulating procoagulant activity of prothrombinase assembled with platelet FV. This platelet FV-platelet TFPIα interaction highlights the important biological activity of pro and anticoagulant proteins located within platelets in regulating hemostatic balance during murine development and into adulthood. DisclosuresMast:Novo Nordisk: Research Funding.

Methylation silencing and reactivation of exogenous genes in lentivirus-mediated transgenic mice.

Taking advantage of their ability to integrate their genomes into the host genome, lentiviruses have been used to rapidly produce transgenic mice in biomedical research. In most cases, transgenes delivered by lentiviral vectors have resisted silencing mediated by epigenetic modifications in mice. However, some studies revealed that methylation caused decreased transgene expression in mice. Therefore, there is conflicting evidence regarding the methylation-induced silencing of transgenes delivered by lentiviral transduction in mice. In this study, we present evidence that the human TTR transgene was silenced by DNA methylation in the liver of a transgenic mouse model generated by lentiviral transduction. The density of methylation on the transgene was increased during reproduction, and the expression of the transgene was completely silenced in mice of the F2 generation. Interestingly, 5-azacytidine (5-AzaC), a methyltransferase inhibitor, potently reactivated the silenced genes in neonatal mice whose hepatocytes were actively proliferating and led to stable transgene expression during development. However, 5-AzaC did not rescue liver transgene expression when administered to adult mice. Moreover, 5-AzaC at the given dose had low developmental toxicity in the newborn mice. In summary, we demonstrate the methylation-induced silencing of an exogenous gene in the liver of a mouse model generated by lentiviral transduction and show that the silenced transgene can be safely and efficiently reactivated by 5-AzaC treatment, providing an alternative way to obtain progeny with stable transgene expression in the case of the methylation of exogenous genes in transgenic mice generated by lentiviral transduction.

Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound–mediated transgene expression in liver

Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50μg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model.

Intramuscular administration of AAV overcomes pre-existing neutralizing antibodies in rhesus macaques

The seroprevalence of neutralizing antibodies (NAbs) to adeno-associated viral (AAV) vector capsids may preclude a percentage of the population from receiving gene therapy, particularly following systemic vector administration. We hypothesized that the use of intramuscular (IM) administration of AAV vectors might circumvent this issue. IM injections were used to administer AAV8 vectors expressing either secreted or non-secreted transgenes into mice and the influence of NAbs supplied by pre-administration of pooled human IgG on transgene expression was evaluated. We then studied the impact of naturally occurring pre-existing AAV8 NAbs on expression of a secreted transgene following IM vector delivery in rhesus macaques. Finally, we evaluated the ability to readminister AAV vectors via IM injections in rhesus macaques. In mice, the presence of AAV8 NAbs had no effect on gene expression in the injected skeletal muscle. However, liver transgene expression following hepatic distribution of the vector was ablated. In rhesus macaques, naturally occurring pre-existing AAV8 NAb titers of ⩽1:160 had no effect on expression levels of a secreted transgene after IM delivery of the vector. Additionally, readministration of AAV vectors was possible by IM injection into the previously injected muscle groups, with no effect on transgene expression by the original vector. Therefore, the presence of pre-existing NAbs in the human population should not preclude subjects from receiving gene therapy by IM administration of the vector so long as sufficient levels of secreted transgene expression can be produced without the involvement of liver.

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.

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo

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. 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. 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. These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

K137R Mutation on Adeno-Associated Viral Capsids Had Minimal Effect on Enhancing Gene DeliveryIn Vivo

The adeno-associated viral (AAV) vector has emerged as an attractive vector for gene therapy applications. Development of AAV vectors with enhanced gene transduction efficiency is important to ease the burden of AAV production and minimize potential immune responses. Rational mutations on AAV capsids have gained attention as a simple method of enhancing AAV transduction efficiency. A single-amino acid mutation, K137R, on AAV1 and AAV8 was recently reported to increase liver transgene expression by 5-10-fold. To determine whether the same mutation on other AAV serotypes would result in similar gene enhancement effects, K137R mutants were generated on AAV7, AAV8, and AAV9, and their effects were evaluated in vivo. Two reporter genes were utilized: the nuclear LacZ gene driven by the cytomegalovirus promoter and the luciferase gene driven by the CB promoter. Surprisingly, we found no difference in luciferase gene expression in the liver or other tissues using either the wild-type AAV8 capsid or AAV8-K137R. LacZ gene expression in the liver by AAV8-K137R was about onefold higher than that of wild-type AAV8. However, no difference was found in other tissues, such as skeletal muscle and cardiac muscle. In addition, no difference was found in transgene expression with either AAV7-K137R or AAV9-K137R mutants. Our results indicated that the K137R mutation on AAV7, AAV8, and AAV9 had minimal to no effect on transduction efficiency in vivo.

Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach

Due to many favourable attributes adenoviruses (Ads) are the most extensively used vectors for clinical gene therapy applications. However, following intravascular administration, the safety and efficacy of Ad vectors are hampered by the strong hepatic tropism and induction of a potent immune response. Such effects are determined by a range of complex interactions including those with neutralising antibodies, blood cells and factors, as well as binding to native cellular receptors (coxsackie adenovirus receptor (CAR), integrins). Once in the bloodstream, coagulation factor X (FX) has a pivotal role in determining Ad liver transduction and viral immune recognition. Due to difficulties in generating a vector devoid of multiple receptor binding motifs, we hypothesised that a small molecule inhibitor would be of value. Here, a pharmacological approach was implemented to block adenovirus transduction pathways. We developed a high throughput screening (HTS) platform to identify small molecule inhibitors of FX-mediated Ad5 gene transfer. Using an in vitro fluorescence and cell-based HTS, we evaluated 10,240 small molecules. Following sequential rounds of screening, three compounds, T5424837, T5550585 and T5660138 were identified that ablated FX-mediated Ad5 transduction with low micromolar potency. The candidate molecules possessed common structural features and formed part of the one pharmacophore model. Focused, mini-libraries were generated with structurally related molecules and in vitro screening revealed novel hits with similar or improved efficacy. The compounds did not interfere with Ad5:FX engagement but acted at a subsequent step by blocking efficient intracellular transport of the virus. In vivo, T5660138 and its closely related analogue T5660136 significantly reduced Ad5 liver transgene expression at 48h post-intravenous administration of a high viral dose (1×1011vp/mouse). Therefore, this study identifies novel and potent small molecule inhibitors of the Ad5 transduction which may have applications in the Ad gene therapy setting.

Systemic TNFα Gene Therapy Synergizes With Liposomal Doxorubicine in the Treatment of Metastatic Cancer

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Viral Transgene Expression Delivered by Repeat Intraocular Adenoviral Vector Injection: in Vivo Live Imaging Study

We delivered adenovirus vector (Ad) via intravitreous injection and monitored transgene (luciferase) expression in living mice (BALB/c) at multiple time points. In vivo live imaging technology was able to assess dynamically intraocular luciferase expression in a single animal population throughout the entire experiment period. Using this information, we were able to determine the optimal time point for readministration of Ad into the eyes and to dynamically study the time course of expression of a second Ad administration. Optical imaging demonstrated the limited period of transgene expression in eyes. Significant transgene signal was also detected in livers. The repeat intraocular delivery of the adenovirus resulted in significant blunting of transgene expression in both eyes and livers compared to the initial delivery. Periocular corticosteroid (triamcinolone acetonide) injection combined with initial Ad delivery was effective to rescue luciferase expression on repeat Ad vector delivery. However, this effect was not observed when corticosteroid was combined with repeat Ad delivery. Although corticosteroid enhanced ocular transgene expression, it also increased transgene expression in liver, which has potential safety implications. This dynamic transgene expression in eyes was successfully traced and monitored via a live imaging technique.

Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates

BackgroundAdeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this work, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression.MethodsThree female Macaca fascicularis were intravenously injected with 1x1013 genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5x1012 genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression.ResultsMacaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system.ConclusionsThese results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.

PTU-043 Development of a liver-specific, tumour-selective recombinant adeno-associated virus vector to target hepatocellular carcinoma

IntroductionHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is currently one of the most difficult to treat. Surgical resection and liver transplantation are considered curative therapies but...

265 DEVELOPMENT OF A LIVER-SPECIFIC, TUMOUR-SELECTIVE RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR TO TARGET HEPATOCELLULAR CARCINOMA

Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is currently one of the most difficult to treat. Surgical resection and liver transplantation are considered curative therapies but they are feasible for only a small number of patients. Other therapeutic options, such as radiofrequency ablation and arterial chemoembolization, are effective only in small tumours. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for gene transfer-based therapeutic approaches for HCC because of their safety profile and remarkable tropism for the liver, with >90% of the vector particles being detectable in hepatocytes following a single systemic administration in non-human primates. Methods Sequence complementarity to miR-122a (122aT) was cloned downstream of luciferase under the control of a new liver specific promoter/enhancer element, HLP, and flanked by the AAV-inverted-terminal-repeats (ITRs). AAV8 capsid-pseudotyped AAV8-HLP-LUC±122aT vector stocks were prepared by 293T-based three-plasmid transient transfection, and used for: in vitro transduction of HCC cell lines HUH-7 (miR-122a-positive) and SNU-387 (miR-122a-negative); in vivo tail vein injection of tumour-bearing SCID and B6 mice. Results A single tail-vein injection of rAAV8 in tumour-bearing mice resulted in selective transduction of the liver as well as of HCC, but not neuroblastoma xenografts. We cloned the 122aT into our expression cassette to improve the transgene expression for HCC, taking advantage of the differential expression of miR-122a (abundant in normal liver but down regulated in most HCC). Normalised transgene expression with AAV8-HLP-LUC-122aT was at least 26.5-fold higher in SNU-387 when compared to HUH-7 cell lines. Tail-vein injection of rAAV8-HLP-LUC resulted in high transgene expression in liver, but mice transduced with rAAV8-HLP-Luc-122aT had little or no luciferase expression despite the proviral DNA presence. Subcutaneous HCC xenografts in mice showed a strong luciferase signal following tail-vein injection of both of these vectors. Conclusion Inclusion of 122aT in the expression cassette allows regulation of rAAV mediated transgene expression in HCC cell lines and normal liver, and reduces expression in miR-122a positive cells (eg, normal hepatocytes). This novel vector, therefore, has the potential to deliver therapeutic transgenes to HCC in preference to normal hepatocytes in the liver, thus limiting toxicity. Competing interests None declared.

A New Method to Determine Antigen-Specific CD8+ T Cell Activity in Vivo by Hydrodynamic Injection.

Hydrodynamic tail vein (HTV) delivery is a simple and rapid tail vein injection method of a high volume of naked plasmid DNA resulting in high levels of foreign gene expression in organs, especially the liver. Compared to other organs, HTV delivery results in more than a 1000-fold higher transgene expression in liver. After being bitten by malaria-infected mosquitoes, malaria parasites transiently infect the host liver and form the liver stages. The liver stages are known to be the key target for CD8+ T cells that mediate protective anti-malaria immunity in an animal model. Therefore, in this study, we utilized the HTV delivery technique as a tool to determine the in vivo cytotoxic effect of malaria antigen-specific CD8+ T cells. Two weeks after mice were immunized with recombinant adenoviruses expressing malarial antigens, the immunized mice as well as naïve mice were challenged by HTV delivery of naked plasmid DNA co-encoding respective antigen together with luciferase using dual promoters. Three days after the HTV challenge, non-invasive whole-body bioluminescent imaging was performed. The images demonstrate in vivo activity of CD8+ T cells against malaria antigen-expressing cells in liver.

Sustained correction of OTC deficiency in spf( ash) mice using optimized self-complementary AAV2/8 vectors.

Ornithine transcarbamylase deficiency (OTCD) is the most common inborn error of urea synthesis. Complete OTCD can result in hyperammonemic coma in the neonatal period which can rapidly become fatal. Current acute therapy involves dialysis; chronic therapy involves the stimulation of alternate nitrogen clearance pathways; and the only curative approach is liver transplantation. AAV vector based gene therapy would add to current treatment options provided the vector delivers high level and stable transgene expression in liver without dose limiting toxicity. In this study, we employed an AAV2/8-based self-complementary (sc) vector expressing the murine OTC gene under a liver-specific thyroxine-binding globulin (TBG) promoter and examined the therapeutic effects in a mouse model of OTCD, the spf ash mouse. Seven days after a single intravenous injection of vector, treated mice showed complete normalization of urinary orotic acid, a measure of OTC activity. We further improved vector efficacy by incorporating a Kozak or Kozak-like sequence into mOTC cDNA which increased the OTC activity by 5- or 2-fold and achieved sustained correction of orotic aciduria for up to 7 months. Our results demonstrate that vector optimizations can significantly improve the efficacy of gene therapy.

Sustained, high transgene expression in liver with plasmid vectors using optimized promoter‐enhancer combinations

Plasmid-based gene therapy approaches often lack long-term transgene expression in vivo as a result of silencing or loss of the vector. One way to overcome these limitations is to combine nonsilenced promoters with strong enhancers. In the present study, we combine murine or human cytomegalovirus (CMV)-derived enhancer elements with the human elongation factor 1α (EF1α) promoter in a plasmid backbone devoid of potentially immunostimulating cytosine-guanine repeat sequences. Luciferase transgene activity was monitored in mouse liver after hydrodynamic plasmid delivery. Luciferase activity of a CMV-promoter driven plasmid rapidly declined within days, whereas the activity of the EF1α driven plasmid remained high for 2 weeks (murine enhancer) and detectable for > 80 days (human enhancer). Expression levels clearly correlated with higher plasmid copy number found in the liver at 2 months after gene delivery. Furthermore, we developed a novel synthetic CMV-EF1α hybrid promoter (SCEP) combining the high activity of CMV and sustained activity of EF1α promoter. The SCEP led to a constitutive three-fold increase in expression levels compared to the EF1α promoter in vivo. This novel combination of enhancer and promoter element with optimized plasmid backbones will pave the way for more efficient nonviral approaches in gene therapy.