Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

The loss of transgene expression has been a major obstacle to the development of nonviral vectors for the treatment of human diseases. We previously demonstrated that bacterial DNA linked to a mammalian expression cassette resulted in transcriptional silencing of the transgene in vivo. To confirm these studies and develop a means to produce a robust DNA vector that is not silenced in vivo, we developed a phage phiC31 integrase-mediated intramolecular recombination technology to prepare minicircle vector DNA devoid of the bacterial backbone and then compared the transgene expression profile of the minicircle with different molecular forms of plasmid DNAs in mice. We demonstrate that minicircular DNAs devoid of bacterial sequences expressed 45- and 560-fold more serum human factor IX and alpha1-antitrypsin, respectively, compared to standard plasmid DNAs transfected into mouse liver. Our data suggest that minicircles are capable of expressing high and persistent levels of therapeutic products in vivo and have a great potential to serve as episomal vectors for the treatment of a wide variety of diseases.

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.

We have developed a new helper-dependent (HD) adenoviral vector FTC that contains 3 cis-acting sequences as stuffer DNA: a human fragment of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. To determine the most robust human coagulation factor IX (hFIX) expression cassette in an adenovirus, we first tested different hFIX expression sequences with or without flanking MARs in first-generation adenoviral vectors. After intravenous infusion of the vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct comparison, a first-generation and a genedeleted vector with identical hFIX expression cassettes were constructed. Both first-generation and HD adenovirustreated animals demonstrated a threshold effect in a dose-response study. With the administration of 2 ؋ 10 9 transducing particles of either vector, supraphysiological serum levels of hFIX were obtained, with the highest expression (41 000 ng/ mL) occurring during the first 2 months after injection. The serum factor IX concentrations, while remaining in the therapeutic range, slowly declined by 95% over a period of 1 year. At this dose, interleukin-6 and tumor necrosis factor-␣ serum concentrations were elevated in animals that received the first-generation but not the HD vector. This study compares the properties of a gene-deleted and first-generation adenovirus with equivalent expression cassettes and suggests that the cis-DNA elements contained in the vector and expression cassette have important effects on gene expression in vivo. (Blood. 2002;99: 3923-3930)

Adoptive cell therapy holds much promise in the treatment of cancer but results in solid tumors have been modest. The notable exception is tumor-infiltrating lymphocyte (TIL) therapy of melanoma, but this approach only works with high-dose preconditioning chemotherapy and systemic interleukin (IL)-2 postconditioning, both of which are associated with toxicities. To improve and broaden the applicability of adoptive cell transfer, we constructed oncolytic adenoviruses coding for human IL-2 (hIL2), tumor necrosis factor alpha (TNF-α), or both. The viruses showed potent antitumor efficacy against human tumors in immunocompromised severe combined immunodeficiency (SCID) mice. In immunocompetent Syrian hamsters, we combined the viruses with TIL transfer and were able to cure 100% of the animals. Cured animals were protected against tumor re-challenge, indicating a memory response. Arming with IL-2 and TNF-α increased the frequency of both CD4+ and CD8+ TILs in vivo and augmented splenocyte proliferation ex vivo, suggesting that the cytokines were important for T cell persistence and proliferation. Cytokine expression was limited to tumors and treatment-related signs of systemic toxicity were absent, suggesting safety. To conclude, cytokine-armed oncolytic adenoviruses enhanced adoptive cell therapy by favorable alteration of the tumor microenvironment. A clinical trial is in progress to study the utility of Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (TILT-123) in human patients with cancer.

• Mobilized hematopoietic stem cells transduced with IV injected HD-Ad5/35 11 vectors home to the BM persist long term.• Our approach allows for the stable genetic modification of primitive, long-term persisting HSPCs.Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35 11 ) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34 1 cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin 2 Sca1 1 Kit 2 cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy. (Blood. 2016;128(18):2206-2217

Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.

Although effective gene therapy vectors have been developed for organ systems such as the liver, an effective delivery vector to the pancreas in vivo has remained elusive. Of the currently available viral vectors, adenovirus and adeno-associated virus (AAV) are two of the most efficient at transducing nondividing cells. We have constructed recombinant adenovirus (AdVLacZ), adeno-associated virus serotype 2 (AAV2LacZ), and pseudotyped adeno-associated virus serotype 5 and 8 (AAV5LacZ, AAV8LacZ) carrying the LacZ reporter, and compared the transduction efficiency of these four vectors in the pancreas of mice in vivo. We showed that adenovirus, AAV2, and AAV8 are capable of transducing the pancreas in vivo, but with different expression kinetics, efficiencies of transduction, and persistence. AdVLacZ-transduced pancreas exhibited maximum LacZ expression at 1 week postdelivery, with greater than 90% of expression lost at 4 weeks. AAV2LacZ-transduced pancreas displayed peak LacZ levels at 4 weeks postdelivery, with no significant decrease in expression for up to 8 weeks. AAV8LacZ was at least 10-fold more efficient than AAV2LacZ in transducing the pancreas in vivo, with significant levels of expression detectable at 1 week, whereas AAV5LacZ did not result in any detectable transgene expression at all tested time points. All three vectors primarily transduced pancreatic acinar cell types, with limited transduction of pancreatic endocrine cells. AdVLacZ elicited a significant leukocyte infiltration early after delivery into the pancreas, whereas none of the AAV vectors elicited a significant leukocyte response. None of the tested vectors caused significant changes in serum amylase or blood glucose levels, suggesting that they do not significantly alter pancreatic function. These vectors will be useful for studying novel gene delivery based treatments in animal models for diabetes and other pancreatic disorders. 405