Showing papers in "Human Gene Therapy in 2010"

Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors.

Abstract: Adeno-associated viruses (AAVs) are small, nonenveloped single-stranded DNA viruses that require helper viruses to facilitate efficient replication. Despite the presence of humoral responses to the wild-type AAV in humans, AAV remains one of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Characterization of the IgG subclass responses to AAV and study of the prevalence of both IgG and neutralizing factors to AAV types 1, 2, 5, 6, 8, and 9 in the human population are of importance for the development of new strategies to overcome these immune responses. Natural exposure to AAV types 1, 2, 5, 6, 8, and 9 can result in the production of antibodies from all four IgG subclasses, with a predominant IgG1 response and low IgG2, IgG3, and IgG4 responses. Prevalences of anti-AAV1 and -AAV2 total IgG determined by enzyme-linked immunosorbent assay were higher (67 and 72%) than those of anti-AAV5 (40%), anti-AAV6 (46%), anti-AAV8 (38%), and anti-AAV9 (47%). Furthermore, data showed that cross-reactions are important. The two highest neutralizing factor seroprevalences were observed for AAV2 (59%) and AAV1 (50.5%) and the lowest were observed for AAV8 (19%) and AAV5 (3.2%). Vectors based on AAV5, AAV8, and AAV9 may have an advantage for gene therapy in humans. Furthermore, among individuals seropositive for AAV5, AAV8, and AAV9, about 70-100% present low titers. Better characterization of the preexisting humoral responses to the AAV capsid and cross-reactivity will allow development of new strategies to circumvent AAV acquired immune responses.

Bone marrow mesenchymal stem cells: historical overview and concepts.

Abstract: Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes. In this review, Dr. Pierre Charbord discusses the historical emergence of the concept of bone marrow MSCs and their possible risks and benefits within the context of future clinical applications.

The regulation of differentiation in mesenchymal stem cells.

Abstract: Mesenchymal stromal/stem cells (MSCs) are a population of stromal cells present in the bone marrow and most connective tissues, capable of differentiation into mesenchymal tissues such as bone and cartilage. MSCs are attractive candidates for biological cell-based tissue repair approaches because of their extensive proliferative ability in culture while retaining their mesenchymal multilineage differentiation potential. In addition to its undoubted scientific interest, the prospect of monitoring and controlling MSC differentiation is a crucial regulatory and clinical requirement. Hence, the molecular regulation of MSC differentiation has been extensively studied. Most of the studies are in vitro, because the identity of MSCs in their tissues of origin in vivo remains undefined. This review addresses the current knowledge of the molecular basis of differentiation of cultured MSCs, with a particular focus on chondrogenesis and osteogenesis. Building on the information coming from developmental biology studies of embryonic skeletogenesis, several signaling pathways and transcription factors have been investigated and shown to play critical roles in MSC differentiation. In particular, the Wnt and transforming growth factor-β/bone morphogenetic protein signaling pathways are well known to modulate in MSCs the molecular differentiation into cartilage and bone. Relevant to the emerging concept of stem cell niches is the demonstration that physical factors can also participate in the regulation of MSC differentiation. Knowledge of the regulation of MSC differentiation will be critical in the design of three-dimensional culture systems and bioreactors for automated bioprocessing through mathematical models applied to systems biology and network science.

Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

Abstract: Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV–adenovirus, AAV–herpesvirus, and AAV–baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotyp...

Immunological aspects of allogeneic mesenchymal stem cell therapies.

Abstract: Allogeneic mesenchymal stem or stromal cells (MSCs) are proposed as cell therapies for degenerative, inflammatory, and autoimmune diseases. The feasibility of allogeneic MSC therapies rests heavily on the concept that these cells avoid or actively suppress the immunological responses that cause rejection of most allogeneic cells and tissues. In this article the validity of the immune privileged status of allogeneic MSCs is explored in the context of recent literature. Current data that provide the mechanistic basis for immune modulation by MSCs are reviewed with particular attention to how MSCs modify the triggering and effector functions of innate and adaptive immunity. The ability of MSCs to induce regulatory dendritic and T-cell populations is discussed with regard to cell therapy for autoimmune disease. Finally, we examine the evidence for and against the immune privileged status of allogeneic MSCs in vivo. Allogeneic MSCs emerge as cells that are responsive to local signals and exert wide-ranging, predominantly suppressive, effects on innate and adaptive immunity. Nonetheless, these cells also retain a degree of immunogenicity in some circumstances that may limit MSC longevity and attenuate their beneficial effects. Ultimately successful allogeneic cell therapies will rely on an improved understanding of the parameters of MSC-immune system interactions in vivo.

4-1BB and CD28 signaling plays a synergistic role in redirecting umbilical cord blood T cells against B-cell malignancies.

Abstract: Umbilical cord blood (UCB) T cells can be redirected to kill leukemia and lymphoma cells by engineering with a single-chain chimeric antigen receptor (CAR) and thus may have general applications in adoptive cell therapy. However, the role of costimulatory molecules in UCB T-cell activation and effector functions in context with CAR remains elusive. To investigate the effect of costimulatory molecules (4-1BB and CD28) on UCB T cells, we transduced UCB T cells with lentiviral vectors expressing Green Fluorescent Protein (GFP) and CAR for CD19 containing an intracellular domain of the CD3ζ chain and either a 4-1BB (UCB-19BBζ) or a CD28 intracellular domain (UCB-1928ζ), both (UCB-1928BBζ), or neither (UCB-19ζ). We found that UCB-19BBζ and UCB-28BBζ T cells exhibited more cytotoxicity to CD19+ leukemia and lymphoma cell lines than UCB-19ζ and UCB-1928ζ, although differences in secretion of interleukin-2 and interferon-γ by these T cells were not evident. In vivo adoptive transfer of these T cells into...

"Mesenchymal" stem cells in human bone marrow (skeletal stem cells): a critical discussion of their nature, identity, and significance in incurable skeletal disease.

Abstract: At the turn of a decade of intensive wishful thinking, "mesenchymal stem cells" are changing their profile, while retaining their charm. As hopes to turn bone into brain or vice versa seem on the wane, we learn (1) that the archetypal "mesenchymal stem cell," the skeletal stem cell found in the bone marrow, can be directly identified as a specialized type of mural cell/pericyte, found in the wall of sinusoids and long known as adventitial reticular cells; (2) that bone marrow skeletal stem cells are also defined by expression of CD146, and can self-renew in vivo, while giving rise to skeletal tissues, and therefore earn consideration as bona fide stem cells; (3) that a broader class of microvascular mural cells endowed with clonogenicity and progenitor properties may exist in other tissues, although their true potency needs to be firmly established by stringent assays and thorough comparisons across tissues; (4) that bone marrow skeletal stem cells display unique angiopoietic and hematopoietic niche-related functions, consisting in their ability to transfer the hematopoietic microenvironment and to guide the assembly of microvascular networks, which seem to define their inherent biology; and (5) that use of skeletal stem cells as disease models, and as models of high-risk strategies for cell and gene therapy specifically in incurable skeletal diseases, may provide new challenges for the next decade, and perhaps reward for medicine in the one that follows.

piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies.

Abstract: In this report, transposition with the piggyBac system is used to introduce a chimeric antigen receptor (CAR) into T lymphocytes. The CD19-specific CAR is expressed long term in expanding T cell populations from its integrated locus and leads to CD19-dependent killing of tumor targets. Chromosomal analysis indicated no signs of genotoxicity.

Restoration of Cone Vision in the CNGA3(-/-) Mouse Model of Congenital Complete Lack of Cone Photoreceptor Function

Abstract: Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3(-/-) mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3(-/-) mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3(-/-) mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.

Genetic Engineering of Mesenchymal Stem Cells and Its Application in Human Disease Therapy

Abstract: The use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed.

Phase I/II study of oncolytic herpes simplex virus NV1020 in patients with extensively pretreated refractory colorectal cancer metastatic to the liver.

Abstract: Geevarghese et al. report results from a phase I/II multicenter study evaluating the safety, pharmacokinetics, and antitumor effects of repeated doses of NV1020, a genetically engineered oncolytic herpes simplex virus, in patients with advanced metastatic colorectal cancer (mCRC). The authors reveal that NV1020 stabilizes liver metastases with minimal toxicity in mCRC and leads to a one-year survival rate of 47.2%.

Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing

Abstract: Vectors based on adeno-associated virus (AAV) are the subject of increasing interest as research tools and agents for in vivo gene therapy. A current limitation on the technology is the versatile and scalable manufacturing of vector. On the basis of experience with AAV2-based vectors, which remain strongly cell associated, AAV vector particles are commonly harvested from cell lysates, and must be extensively purified for use. We report here that vectors based on other AAV serotypes, including AAV1, AAV8, and AAV9, are found in abundance in, and can be harvested from, the medium of production cultures carried out with or without serum. For AAV2, this difference in compartmentalization is largely due to the affinity of the AAV2 particle for heparin, because an AAV2 variant in which the heparin-binding motif has been ablated gives higher yields and is efficiently released from cells. Vector particles isolated from the culture medium appear to be functionally equivalent to those purified from cell lysates in terms of transduction efficiency in vitro and in vivo, immunogenicity, and tissue tropism. Our findings will directly lead to methods for increasing vector yields and simplifying production processes for AAV vectors, which should facilitate laboratory-scale preparation and large-scale manufacture.

Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.

Abstract: A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 918 x 10¹¹ particles/ml; 95% confidence interval [CI], 789 x 10¹¹ to 105 x 10¹² particles/ml), vector genomes ({X}, 328 x 10¹⁰ vector genomes/ml; 95% CI, 270 x 10¹⁰ to 475 x 10¹⁰ vector genomes/ml), transducing units ({X}, 509 x 10⁸ transducing units/ml; 95% CI, 200 x 10⁸ to 960 x 10⁸ transducing units/ml), and infectious units ({X}, 437 x 10⁹ TCID₅₀ IU/ml; 95% CI, 206 x 10⁹ to 926 x 10⁹ TCID₅₀ IU/ml) Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94% One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards Anticipated uses of the rAAV2 RSM are discussed

Tat-BMPs-PAMAM conjugates enhance therapeutic effect of small interference RNA on U251 glioma cells in vitro and in vivo.

Abstract: Han and colleagues engineer an existing nonviral biocarrier based on polyamidoamine dendrimers to deliver an interfering RNA to downregulate expression of the oncogenic epidermal growth factor receptor in gliomas. To enable cell targeting and entry, and eventual transduction, portions of the HIV-1 Tat protein and magnetic nanoparticle technology were incorporated in the design.

Adeno-Associated Virus Serotype 6 Capsid Tyrosine-to-Phenylalanine Mutations Improve Gene Transfer to Skeletal Muscle

Abstract: Adeno-associated viral (AAV) vectors are the most efficient in vivo gene transfer tools for gene therapy applications. Efforts have been made to translate encouraging results in small animal models to human patients. However, the need for large quantities of vector for clinical application remains a great challenge. Developing novel AAV vectors with enhanced infectivity may reduce the high vector dose requirement for many applications such as gene therapy for muscular dystrophy. Selective mutation of AAV capsid surface-exposed tyrosine (Y) is a novel strategy to improve transduction efficiency. AAV6 has been considered one of the most robust muscle gene delivery vehicles. Here, we hypothesize that AAV6 transduction efficiency can be further enhanced by mutating surface Y to phenylalanine (F). We found that mutants AAV6-Y445F and AAV6-Y731F, especially the former, achieved more efficient gene transfer than the original AAV6 after intramuscular administration to mice. Expression of both firefly luciferase and alkaline phosphatase reporter genes increased up to 8-fold and DNA copy numbers in muscle increased up to 6-fold. Our results suggest that tyrosine-mutant AAV6 vectors may represent powerful tools for testing muscle gene therapy in animal models and potentially in humans.

Interleukin-10 Delivery via Mesenchymal Stem Cells: A Novel Gene Therapy Approach to Prevent Lung Ischemia–Reperfusion Injury

Abstract: Ischemia reperfusion (IR)-induced lung injury is a major cause of primary graft failure after lung transplantation. In this study, Manning and colleagues examine whether bone-marrow derived mesenchymal stem cells engineered to secrete IL-10 can prevent lung IR injury in a rat model.

Human hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus serotype 3

Abstract: Adeno-associated viruses (AAVs) use a variety of cellular receptors/coreceptors to gain entry into cells. A number of AAV serotypes are now available, and the cognate receptors/coreceptors for only a handful of those have been identified thus far. Of the 10 commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells in general. However, in our more recent studies, we observed that AAV3 vectors transduced human liver cancer cells remarkably well, which led to the hypothesis that AAV3 uses hepatocyte growth factor receptor (HGFR) as a cellular coreceptor for viral entry. AAV3 infection of human liver cancer cell lines was strongly inhibited by hepatocyte growth factor, HGFR-specific small interfering RNA, and anti-HGFR antibody, which corroborated this hypothesis. However, AAV3 vectors failed to transduce murine hepatocytes, both in vitro and in vivo, suggesting that AAV3 specifically uses human HGFR, but not murine HGFR, as a cellular coreceptor for transduction. AAV3 may prove to be a useful vector for targeting human liver cancers for the potential gene therapy.

Electrically mediated delivery of plasmid DNA to the skin, using a multielectrode array.

Abstract: The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take full advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is a strong candidate to meet this delivery criterion. Electroporation of the skin is a simple, direct, in vivo method to deliver genes for therapy. Previously, delivery to the skin was performed by means of applicators with relatively large distances between electrodes, resulting in significant muscle stimulation and pain. These applicators also had limitations in controlling the directionality of the applied field. To resolve this issue, a system consisting of an array of electrodes that decreased the distance between them and that were independently addressable for directional control of the field was developed. This new multielectrode array (MEA) was compared with an established electrode. In a rat model, comparable reporter expression was seen after delivery with each electrode. Delivery was also evaluated in a guinea pig model to determine the potential of this approach in an animal model with skin thickness and structure similar to human skin. The results clearly showed that effective delivery was related to both the electrode and the parameters chosen. With the MEA, the muscle twitching associated with application of electric fields was notably reduced compared with conventional electrode systems. This is important, as it will facilitate the translation of electroporation-mediated gene delivery to skin for clinical use with DNA vaccines or for therapies for cancer or protein deficiencies.

Long-Term Type VII Collagen Restoration to Human Epidermolysis Bullosa Skin Tissue

Abstract: In spite of advances in the molecular diagnosis of recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disease due to a deficiency of type VII collagen at the basement membrane zone (BMZ) of stratified epithelium, current therapy is limited to supportive palliation. Gene delivery has shown promise in short-term experiments; however, its long-term sustainability through multiple turnover cycles in human tissue has awaited confirmation. To characterize approaches for long-term genetic correction, retroviral vectors were constructed containing long terminal repeat-driven full-length and epitope-tagged COL7A1 cDNA and evaluated for durability of type VII collagen expression and function in RDEB skin tissue regenerated on immune-deficient mice. Type VII collagen expression was maintained for 1 year in vivo, or over 12 epidermal turnover cycles, with no abnormalities in skin morphology or self-renewal. Type VII collagen restoration led to correction of RDEB disease features, including reestablishment of anchoring fibrils at the BMZ. This approach confirms durably corrective and noninjurious gene delivery to long-lived epidermal progenitors and provides the foundation for a human clinical trial of ex vivo gene delivery in RDEB.

Replacement Gene Therapy with a Human RPGRIP1 Sequence Slows Photoreceptor Degeneration in a Murine Model of Leber Congenital Amaurosis

Abstract: RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.

Interference of CD40L-Mediated Tumor Immunotherapy by Oncolytic Vesicular Stomatitis Virus

Abstract: Galivo and colleagues examine the efficacy of two different vector platforms in tumor immunotherapy. The authors report that replication-defective adenovirus (Ad)- and vesicular stomatitis virus (VSV)-based vectors induce antitumor immunity via different therapeutic effector mechanisms.

Evaluation of an Attenuated Vesicular Stomatitis Virus Vector Expressing Interferon-β for Use in Malignant Pleural Mesothelioma: Heterogeneity in Interferon Responsiveness Defines Potential Efficacy

Abstract: Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic To avoid toxicity, a vector was created by introducing the interferon-β (IFN-β) gene (VSVIFN-β) We conducted this study to determine the ability of VSVIFN-β to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro VSVhIFN-β did not cause cytotoxicity in any normal lines Only 4 of 12 lines were effectively lysed by VSVhIFN-β In the eight resistant lines, pretreatment with IFN-β prevented lysis of cells by VSVGFP, and VSV infection or addition of IFN-β protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2′,5′-oligo-adenylate-synthetase (2′5′-OAS) mRNA In the susceptible lines, there was no protection by pretreatment

ACE2 Overexpression Ameliorates Left Ventricular Remodeling and Dysfunction in a Rat Model of Myocardial Infarction

Abstract: The purpose of this study was to test the hypothesis that overexpression of angiotensin-converting enzyme 2 (ACE2) may favorably affect left ventricular (LV) remodeling and function after myocardial infarction (MI). The left anterior descending coronary artery was ligated to produce anterior MI in 100 Wistar-Kyoto rats that were randomly divided into Ad-ACE2, Ad-ACE2+A779, Ad-EGFP, model, and sham groups. Two weeks later, rats in the Ad-ACE2 and Ad-EGFP groups received direct intramyocardial injection of Ad-ACE2 and Ad-EGFP, respectively. Rats in the Ad-ACE2+A779 group received both intramyocardial injection of Ad-ACE2 and a continuous intravenous infusion of A779 for 15 days. LV volume and systolic function, the extent of myocardial fibrosis, and levels of ACE2, angiotensin II (Ang II), and collagen I protein expression were evaluated. Four weeks after ACE2 gene transfer, the Ad-ACE2 group showed reduced LV volume, extent of myocardial fibrosis, and expression levels of ACE, Ang II, and collagen I in the myocardium, and increased LV ejection fraction and levels of ACE2 activity and expression in comparison with the Ad-EGFP and model groups. These results suggest that ACE2 overexpression attenuated LV fibrosis and improved LV remodeling and systolic function. In conclusion, overexpression of ACE2 favorably affected the pathological process of LV remodeling after MI by inhibiting ACE activity, reducing AngII levels, and up-regulating Ang-(1-7) expression, thus providing a potential therapeutic target in the treatment of heart failure.

High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.

Abstract: Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is ∼10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y50...

Mesenchymal stem cells and osteoarthritis: remedy or accomplice?

Abstract: Multipotent mesenchymal stromal or stem cells (MSCs) are likely to be agents of connective tissue homeostasis and repair. Because the hallmark of osteoarthritis (OA) is degeneration and failure to repair connective tissues it is compelling to think that these cells have a role to play in OA. Indeed, MSCs have been implicated in the pathogenesis of OA and, in turn, progression of the disease has been shown to be therapeutically modulated by MSCs. This review discusses current knowledge on the potential of both marrow- and local joint-derived MSCs in OA, the mode of action of the cells, and possible effects of the osteoarthritic niche on the function of MSCs. The use of stem cells for repair of isolated cartilage lesions and strategies for modulation of OA using local cell delivery are discussed as well as therapeutic options for the future to recruit and appropriately activate endogenous progenitors and/or locally systemically administered MSCs in the early stages of the disease. The use of gene therapy protocols, particularly as they pertain to modulation of inflammation associated with the osteoarthritic niche, offer an additional option in the treatment of this chronic disease. In summary, elucidation of the etiology of OA and development of technologies to detect early disease, allied to an increased understanding of the role MSCs in aging and OA, should lead to more targeted and efficacious treatments for this debilitating chronic disease in the future.

A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.

Abstract: Van Pijkeren and colleagues at University College in Cork, Ireland describe a novel regulatable gene transfer platform based on Listeria monocytogenes. Using this system, the authors demonstrate successful in vivo gene delivery of firefly luciferase to tumors in murine models and ex vivo to patient breast tumor samples.

Modification of Integration Site Preferences of an HIV-1-Based Vector by Expression of a Novel Synthetic Protein

Abstract: HIV-1-based lentiviral vectors are a promising tool for gene therapy. However, integration of a lentiviral vector into host cell genes may lead to the development of cancer. Therefore, control of integration site selection is critical to the successful outcome of gene therapy approaches that use these vectors. The discovery that integration site selection by HIV-1 and HIV-1-based vectors is controlled by the LEDGF/p75 protein has presented new opportunities to control integration site selection. In this study, we tested the hypothesis that a fusion protein containing the C-terminal HIV integrase-binding portion of LEDGF/p75, and the N-terminal chromodomain of heterochromatin protein-1α (HP1α), can target HIV-1 vector DNA outside of genes. We show that this fusion protein, termed TIHPLE, associates with the heterochromatin hallmark trimethylated Lys-9 of histone H3 (H3K9me3). Transient overexpression of TIHPLE alters integration site selection by an HIV-1-based vector and decreases the number of integration events that occur in genes. This change in integration site selection was achieved without a reduction in overall integration efficiency. Furthermore, we show that TIHPLE increases integration in the vicinity of H3K9me3 and in repetitive DNA sequences. These data provide a novel approach to address the problem of the tendency of retroviral vectors to integrate at undesirable sites of the human genome.

Safety Studies on Intrahepatic or Intratumoral Injection of Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Rodents and Nonhuman Primates

Abstract: The present study reports on the safety and toxicity of an oncolytic vesicular stomatitis virus (VSV)-based gene therapy in animal models in preparation for clinical trials for the treatment of hepatocellular carcinoma (HCC). To restrict spread of the oncolytic virus beyond the tumor, the VSV vector carries human interferon-β for its potent antiviral effect. The study design used rat and nonhuman primate models, and included an evaluation of safety.

Development of Adoptive Cell Therapy for Cancer: A Clinical Perspective

Abstract: Adoptive cellular therapy provides the promise of a potentially powerful general treatment for cancer. Although this is a complex and challenging field, there have been major advances in basic and translational research resulting in clinical trial activity that is now beginning to confirm this promise. However, these trials are also identifying new challenges and this review focuses on these clinical issues. For tumors such as melanoma, in which tumor-specific T cells can be readily identified and isolated, the adoptive transfer of "tumor-infiltrating lymphocytes" (TILs) already appears to offer significant patient benefit and this approach now warrants further development. Genetically engineered T cells offer a means to endow peripheral blood T cells with antitumor activity and in principle these techniques could allow the treatment of a wide range of cancers. Genetic engineering also offers the means to endow T cells with new properties and enhanced functions. There have been clear proof-of-principle trials showing responses with T cell receptor (TCR)-engineered T cells and this can be built on with further development. By contrast, other trials have produced significant toxicity related to expression of target antigen on normal tissue, particularly with enhanced receptors. The challenge ahead lies in understanding how to achieve the balance between targeted antitumor immune responses while avoiding toxicity associated with on-target destruction of antigen-expressing normal tissues. Cellular therapy of cancer will need continued preclinical evaluation as well as carefully designed clinical trials addressing the various issues. For these challenges to be fully assessed, and for progression to a widely used, effective and safe therapy, development as cooperative groups is an appropriate way forward.

Enhanced Transfection Efficiency into Macrophages and Dendritic Cells by a Combination Method Using Mannosylated Lipoplexes and Bubble Liposomes with Ultrasound Exposure

Abstract: To achieve effective gene therapy, it is necessary to selectively and efficiently transfect therapeutic gene into targeted cells. In this study, we developed a combination method using mannosylated lipoplexes, which show selectivity to antigen-presenting cells such as macrophages and dendritic cells, and bubble liposomes (BLs), which are known to enhance transfection efficiency on exposure to ultrasound (US). In cultured mouse macrophages, known for the expression of mannose receptors, the transfection efficiency of this combination method using mannosylated lipoplexes and BLs with US was higher than that of naked pDNA or unmodified lipoplexes and BLs. In the liver and spleen, the in vivo transfection efficiency of this combination method was significantly higher than that of naked pDNA or unmodified lipoplexes and BLs with US. Transfection in hepatic nonparenchymal cells using this combination method was about 12 times higher than that in hepatic parenchymal cells. As far as splenic transfection...