Gene Therapy and Regulation

Impressive progress has been made in clinical development of adeno-associated virus (AAV) vectors over the last 15 years in more than 40 clinical trials, involving many hundreds of subjects with vector delivery by many different routes and at a wide range of doses. Preclinical expectations, including an excellent safety profile, the tendency to remain at a local delivery site, and the persistence of expression for years after delivery, are being borne out in clinical trials. Immune responses to the vectors in human subjects, less well anticipated by preclinical studies, are now being examined in current AAV vector clinical trials, in order to determine in what circumstances such responses might occur or be problematic. Development of AAV vectors is likely to continue to be an expanding field. The current successes in the subjects with retinal degeneration and Parkinson's disease are highly encouraging for AAV vectors and for the whole field of gene therapy.

Directed evolution through genetic engineering of viral capsids followed by selection has emerged as a powerful means to create novel recombinant adeno-associated virus (rAAV) vectors with desired tropism and enhanced properties. One of the most effective approaches uses rAAV-based random peptide display libraries. Here we report a novel system based on an infection-defective rAAV1.9-3 as a platform for random peptide display, and show that biopanning of the libraries in vitro effectively identifies the peptides that restore and enhance rAAV transduction. rAAV1.9-3 has a genetically engineered AAV1 capsid with amino acids 445–568 being replaced with those of AAV9, and has been identified as a variant exhibiting significantly impaired infectivity and delayed blood clearance when infused into mice. In this study, we generated rAAV1.9-3 variant libraries in which 7- or 12-mer random peptides were expressed at the capsid amino acid position 590. Three rounds of positive selection for primary human dermal fibroblasts successfully identified new rAAV-peptide variants that transduce them more efficiently than the prototype rAAV2. Thus our study demonstrates that an infection-defective rAAV variant serves as a novel detargeted platform for random peptide display libraries. We also describe a brief review of recent progress in rAAV-based random peptide display library approaches.

The cardinal motor symptoms of Parkinson's disease (PD) are associated with the profound depletion of dopamine in the striatum. The replacement of dopamine is the most straightforward strategy to improve motor performance in PD. Researchers have been developing gene therapy aimed at local production of dopamine via the introduction of dopamine-synthesizing enzyme genes into the putamen. Two phase I clinical studies have used recombinant adeno-associated virus (AAV) vectors to transfer the aromatic L-amino acid decarboxylase (AADC) gene into the putamen to restore efficient conversion of orally administered L-3,4-dihydroxyphenylalanine (L-dopa). The initial results of these studies have not only confirmed the safety of AAV vectors, but have also demonstrated the alleviation of motor symptoms associated with PD. Interestingly motor performance in the "off" medication state was improved after gene therapy, suggesting long-term modulation of dopaminergic signals in the striatal neurons was induced by gene transfer. Gene delivery of tyrosine hydroxylase (TH) and guanosine triphosphate cyclohydrolase I (GCH) in addition to AADC may help to avoid motor fluctuations associated with intermittent intake of L-dopa by continuously supplying dopamine in the putamen. A clinical study of such triple gene transfer is presently underway using equine infectious anemia virus (EIAV) vector.

Adoptive immunotherapy with tumor infiltrating lymphocytes (TILs) is a potent therapy for metastatic melanoma. In this process, naturally occurring tumor reactive TILs that bear T-cell receptors (TCR) targeted against tumor cells are generated ex vivo and administrated into the patients. The generation of tumor-reactive T cells is not always possible in all of the patients. To overcome this limitation, we can now insert highly avid TCRs into T cells that can recognize tumor antigens. Genetic engineering of TCR genes into normal T cells is a powerful new strategy to generate large numbers of defined antigen-specific cells for therapeutic application. This approach has evolved beyond experimental stage into a clinical reality. The feasibility of TCR-engineered T cells has been shown to be an effective clinical strategy resulting in the regression of established tumors in recent clinical trials. In this review, we discuss the progress and prospects of TCR-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers.

A challenge to develop adenovirus (Ad)-mediated therapeutics has been issued to treat metastatic cancer via systemic administration. For effective gene therapeutics against primary and metastatic lesions, a systemically injectable tumor-targeting Ad vector system must be developed. Systemic delivery of Ad, however, is hampered by immune response against Ad, short half-life in the circulation, liver uptake, and low accumulation at target disease sites. Modification of the Ad surface allows Ad to circulate in the bloodstream for a longer time, to be not targeted to the liver, and to passively accumulate in tumor sites via enhanced permeation and retention effects. The addition of affinity tags results in active targeting and high efficacy. Strategies including addition of polymer complexes, chemical modifications, and targeting moieties have been applied to develop systemically injectable Ad gene therapeutic carriers. More versatile designs of Ad hybrid complexes have been developed with inorganic nanoparticles, polymers, and lipids, making smart nanomedicine possible. Integration of viral and nonviral nanomaterials can substantially synergize both fields, creating a new concept of gene therapeutics. Here, we describe the various Ad hybrid complex systems used to overcome the limited clinical applicability of conventional Ads and enable effective treatment of distant metastatic tumors via systemic injection.

Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for cancer. We have developed a triple-mutated, third-generation oncolytic HSV-1, G47Δ, by introducing an additional genetic mutation to the viral genome of G207, a second generation HSV-1, used in clinical trials for malignant glioma in the United States. Preclinical studies demonstrated that G47Δ exhibited increased antitumor efficacy in various tumor models while preserving the safety of G207. Prior to the first-in-man clinical trial, G47Δ genome structure analysis, stability tests, and preclinical safety evaluation using HSV-1-susceptible A/J mice were performed. After development and optimization of manufacturing processes, clinical-grade G47Δ was produced at the GMP vector production facility of the University of Tokyo. Quality tests under GLP were completed for clinical products at 4 manufacturing steps. The first clinical trial of G47Δ is designed as an open labeled, single armed, phase I-II study for patients with recurrent glioblastoma. Patients with a single lesion, age 18 or older, and with Karnofsky Performance Scale (KPS) 70% or higher are enrolled. G47Δ is administered stereotactically into multiple sites of the tumor, twice within 14 days. The primary endpoint is to assess the safety of G47Δ, and the secondary endpoint is to assess the efficacy by tumor size and progression-free survival. After 3 years of contact with and review by regulatory authorities, the final governmental approval was obtained in May 2009, and the patient registration began in November 2009. In this paper, we also review the background of the clinical development of G47Δ.

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations of the DMD gene, which encodes a 427-kDa spectrin-like cytoskeletal protein, dystrophin. Exon skipping induced by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene and rescue dystrophin expression. We recently demonstrated that systemic delivery of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) targeting exons 6 and 8 of the canine DMD gene efficiently recovered functional dystrophin at the sarcolemma of dystrophic dogs, and improved performance of the affected dogs without serious side effects. As a strategy to target hot spots of mutation in the DMD gene, we also tried exon 51-skipping using PMOs in mdx52 mice to convert an out-of-frame mutation into an in-frame mutation with restoration of dystrophin expression in various muscles and improvement of pathology and function. Progress in adeno-associated virus vector serotype 9 (AAV-9)-mediated DMD gene therapy has enabled the delivery of the therapeutic gene to the whole musculature, including cardiac muscle, while evoking minimal immunological reactions in mice, dogs, and non-human primates. Furthermore, DMD-derived patient-specific induced pluripotent stem (iPS) cells could be a potential source for cell therapy, although there are at present hurdles to be overcome. In the future, this technology could be used in combination with exon skipping or AAV-mediated gene therapy to achieve clinical benefits.