Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing: Targeting Mobilized CD34+ Cells for Correction of Fabry Disease

Fabry disease is a rare lysosomal storage disorder (LSD). We designed multiple recombinant lentivirus vectors (LVs) and tested their ability to engineer expression of human α-galactosidase A (α-gal A) in transduced Fabry patient CD34+ hematopoietic cells. We further investigated the safety and efficacy of a clinically directed vector, LV/AGA, in both ex vivo cell culture studies and animal models. Fabry mice transplanted with LV/AGA-transduced hematopoietic cells demonstrated α-gal A activity increases and lipid reductions in multiple tissues at 6 months after transplantation. Next we found that LV/AGA-transduced Fabry patient CD34+ hematopoietic cells produced even higher levels of α-gal A activity than normal CD34+ hematopoietic cells. We successfully transduced Fabry patient CD34+ hematopoietic cells with “near-clinical grade” LV/AGA in small-scale cultures and then validated a clinically directed scale-up transduction process in a GMP-compliant cell processing facility. LV-transduced Fabry patient CD34+ hematopoietic cells were subsequently infused into NOD/SCID/Fabry (NSF) mice; α-gal A activity corrections and lipid reductions were observed in several tissues 12 weeks after the xenotransplantation. Additional toxicology studies employing NSF mice xenotransplanted with the therapeutic cell product demonstrated minimal untoward effects. These data supported our successful clinical trial application (CTA) to Health Canada and opening of a “first-in-the-world” gene therapy trial for Fabry disease

Comparison of Schwann Cells Derived From Peripheral Nerve With Schwann Cells Differentiated From Skin-derived Precursors

Schwann cells are the glial cells of the peripheral nervous system. When transplanted into the injured central or peripheral nervous systems they promote repair. Traditionally Schwann cells have been isolated from the sciatic nerve, creating nerve-SC. An alternative Schwann cell source is from the differentiation of skin-derived precursors (SKPs), stem cells found in the skin, to Schwann cells (SKP-SC). SKP-SC have shown enhanced regenerative ability compared to nerve-SC. This study compares nerve-SC with SKP-SC at the functional and gene expression level to determine their degree of similarity and find their sources of variance. The functional ability of both Schwann cell types appeared similar. Their gene expression, as assessed by microarray, was similar but not identical. Genes that differed between nerve-SC and SKP-SC may represent differences important to regeneration. The similarity of SKP-SC to nerve-SC supports the use of SKP-SC for repair, and reasons for enhanced regeneration by SKP-SC are suggested.MAS

Improving diagnosis, understanding, and treatment of Farber disease

Farber disease (FD) is an ultra-rare Lysosomal Storage Disorder. It is caused by mutations in ASAH1, resulting in reduced activity of acid ceramidase and ceramide accumulation. The disease is poorly understood due to its rarity and the often short lifespan of patients. FD is systemic, with prominent hematological and sometimes neurological components. To better understand the disease, I characterized the hematopoietic and neurological effects of FD in the first mouse model, where the ASAH1 patient mutation P362R was knocked-in. Mice with FD have enlarged organs due to the accumulation of Mac-2+, foamy macrophages. This accumulation disrupts the organ architecture of hematopoietic-associated organs, including the BM and thymus, resulting in an almost complete loss of developing B and T cells in these organs, respectively, and an excess of hematopoietic stem and progenitor cells in the bone marrow. In the brain, mice with FD also have excess macrophages/microglia, astrocytosis, and hydrocephaly. To improve diagnosis of FD, I identified a plasma cytokine profile that distinguishes patients with FD from those with a disease that it is commonly misdiagnosed as, Juvenile Idiopathic Arthritis. The most elevated of these cytokines was monocyte chemotactic protein 1, and it alone or with the other elevated cytokines was associated with the presence of FD with 80% accuracy. These cytokines were normalized in FD patients who had received hematopoietic stem cell transplantation (HSCT). Finally, to reduce these signs of FD, I tested the efficacy of HSCT. HSCT from WT mice to FD mice more than doubled their lifespan from 7-13 weeks to a median of 27 weeks and a maximum of 40 weeks. Ceramide levels were normalized, and some peripheral signs of the disease were reduced. While beneficial, HSCT did not improve all symptoms. Through better diagnosis and understanding of FD, more effective treatments can be developed.Ph.D

Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

<div><p>Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated <i>in vitro</i> into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. <i>In vivo</i>, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from <i>ex vivo</i> stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells <i>in vitro</i>, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in <i>in vivo</i> settings. These observations must be considered in drafting any regeneration strategies.</p> </div

Rapamycin treatment down regulates SMA expression of SKPs.

<p>SKPs were cultured in medium containing either EGF/FGF +/− Rapamycin or 15% FBS +/− Rapamycin for one week. A: SMA expression was quantified by qPCR. Graph shows mean +/− SE of n = 4. B: SKPs were cultured in medium containing either EGF/FGF +/− Rapamycin or 3% FBS +/− Rapamycin for one week. SMA expression and S6 phosphorylation was analyzed by IF staining. Representative images of n = 10 are shown.</p

SMC differentiation of SKPs correlates with reduced S6 phosphorylation.

<p>A: SKPs were cultured in medium containing 15% FBS for 20 min. S6 phosphorylation was analyzed by IF staining. Representative images on n = 10 are shown. B: SKPs were exposed to conditioned medium from either stretched or non-stretched bladders for 20 min. S6 phosphorylation was analyzed by IF staining. Fluorescent intensity was quantified by image analysis using Volocity software. Graph represents mean +/− SE of n = 50 cells.</p