In Vivo Gene Therapy for Canine SCID-X1 Using Cocal-Pseudotyped Lentiviral Vector.

Hematopoietic stem and progenitor cell (HSPC)-based ex vivo gene therapy has demonstrated clinical success for X-linked severe combined immunodeficiency (SCID-X1) patients who lack a suitable donor for HSPC transplantation. Nevertheless, this form of treatment is associated with an increased risk of infectious disease complications and genotoxicity mainly due to the conditioning regimen. In addition, ex vivo gene therapy approaches require sophisticated facilities to manufacture gene-modified cells and to care for the patients after chemotherapy. Considering these impediments, we have developed an in vivo gene therapy approach to treat canine SCID-X1 after HSPC mobilization and systemic delivery of the therapeutic vector. Here, we investigated the use of the cocal envelope to pseudotype a lentiviral (LV) vector expressing a functional gammaC gene. The cocal envelope is resistant to serum inactivation compared with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope and thus well suited for systemic delivery. Two SCID-X1 neonatal canines treated with this approach achieved long-term therapeutic immune reconstitution with no prior conditioning. Therapeutic levels of gene-corrected CD3+ T cells were demonstrated for at least 16 months, and all other correlates of T cell functionality were within normal range. Retroviral integration-site analysis demonstrated polyclonal T cell reconstitution. Comparative analysis of integration profiles of foamy viral (FV) vector and cocal LV vector after in vivo gene therapy found distinct integration-site patterns. These data demonstrate that clinically relevant and durable correction of canine SCID-X1 can be achieved with in vivo delivery of cocal LV. Since manufacturing of cocal LV is similar to VSV-G LV, this approach is easily translatable to a clinical setting, thus providing for a highly portable and accessible gene therapy platform for SCID-X1.

Effective Multi-lineage Engraftment in a Mouse Model of Fanconi Anemia Using Non-genotoxic Antibody-Based Conditioning.

Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow, facilitating donor cell engraftment. Although effective, a major issue with chemotherapy is the systemic genotoxicity that increases the risk for secondary malignancies. Antibody conjugates targeting hematopoietic cells are an emerging non-genotoxic method of opening the marrow niche and promoting engraftment of transplanted cells while maintaining intact marrow cellularity. Specifically, this platform would be useful in diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita, Schwachman-Diamond syndrome, and Fanconi anemia (FA). Our approach utilizes antibody-drug conjugates (ADC) as an alternative conditioning regimen in an FA mouse model of autologous transplantation. Antibodies targeting either CD45 or CD117 were conjugated to saporin (SAP), a ribosomal toxin. FANCA knockout mice were conditioned with either CD45-SAP or CD117-SAP prior to receiving whole marrow from a heterozygous healthy donor. Bone marrow and peripheral blood analysis revealed equivalent levels of donor engraftment, with minimal toxicity in ADC-treated groups as compared with cyclophosphamide-treated controls. Our findings suggest ADCs may be an effective conditioning strategy in stem cell transplantation not only for diseases where traditional chemotherapy is not tolerated, but also more broadly for the field of blood and marrow transplantation.

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy.

Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.

Aging: central role for autophagy and the lysosomal degradative system.

The lysosomal network is the major intracellular proteolytic system accounting for more than 98% of long-lived bulk protein degradation and recycling particularly in tissues such as liver and muscles. Lysosomes are the final destination of intracellular damaged structures, identified and sequestered by the processes of macroautophagy and chaperone-mediated autophagy (CMA). In the process of macroautophagy, long-lived proteins and other macromolecular aggregates and damaged intracellular organelles are first engulfed by autophagosomes. Autophagosomes themselves have limited degrading capacity and rely on fusion with lysosomes. Unlike macroautophagy, CMA does not require intermediate vesicle formation and the cytosolic proteins recognized by this pathway are directly translocated to the lysosomal membrane. Aging is a universal phenomenon characterized by progressive deterioration of cells and organs due to accumulation of macromolecular and organelle damage. The continuous removal of worn-out components and replacement with newly synthesized ones ensures cellular homeostasis and delays the aging process. Growing evidence indicate that the rate of autophagosome formation and maturation and the efficiency of autophagosome/lysosome fusion decline with age. In addition, a progressive increase in intralysosomal concentration of free radicals and the age pigment lipofuscin further diminish the efficiency of lysosomal protein degradation. Therefore, integrity of the autophagosomal-lysosomal network appears to be critical in the progression of aging. Discovery of the genes involved in the process of autophagy has provided insight into the various molecular pathways that may be involved in aging and senescence. In this review, we discuss the cellular and molecular mechanisms involved in autophagy and the role of autophagosome/lysosome network in the aging process.

Autophagy in aging and in neurodegenerative disorders.

Autophagy (ATG) is the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles. Cellular homeostasis requires continuous removal of worn-out components and replacement with newly synthesized ones. Studies in yeast and other mammalian systems have increased our knowledge of the molecular mechanism of autophagy and the role of autophagy in various pathological conditions. Discovery of the genes involved in the process of autophagy has provided insight into the involvement of various molecular pathways. Growing evidence has indicated that diminished autophagic activity may play a pivotal role in the aging process. Cellular aging is characterized by a progressive accumulation of nonfunctional cellular components owing to oxidative damage and a decline in turnover rate and housekeeping mechanisms. Lysosomes are key organelles in the aging process due to their involvement in both macroautophagy and other housekeeping mechanisms. Autophagosomes themselves have limited degrading capacity and rely on fusion with lysosomes. Accumulation of defective mitochondria also appears to be critical in the progression of aging. Inefficient removal of nonfunctional mitochondria by lysosomes constitutes a major issue in the aging process. Autophagy has been associated with a growing number of pathological conditions, including cancer, myopathies, and neurodegenerative disorders. In this review, we discuss the cellular and molecular mechanisms involved in autophagy, the mechanisms of aging, and the possible role of autophagy in this process. Understanding the mechanisms by which autophagy impacts aging may provide useful molecular targets for pharmaceuticals designed to delay aging or correct conditions of premature aging.