Lentivirus-mediated gene therapy for Fabry disease

Enzyme and chaperone therapies are used to treat Fabry disease. Such treatments are expensive and require intrusive biweekly infusions; they are also not particularly efficacious. In this pilot, single-arm study (NCT02800070), five adult males with Type 1 (classical) phenotype Fabry disease were infused with autologous lentivirus-transduced, CD34+-selected, hematopoietic stem/progenitor cells engineered to express alpha-galactosidase A (α-gal A). Safety and toxicity are the primary endpoints. The non-myeloablative preparative regimen consisted of intravenous melphalan. No serious adverse events (AEs) are attributable to the investigational product. All patients produced α-gal A to near normal levels within one week. Vector is detected in peripheral blood and bone marrow cells, plasma and leukocytes demonstrate α-gal A activity within or above the reference range, and reductions in plasma and urine globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are seen. While the study and evaluations are still ongoing, the first patient is nearly three years post-infusion. Three patients have elected to discontinue enzyme therapy

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

Integrin α3 β1 : cancer-associated glycans and colon cancer

Colon cancer arises from the gradual accumulation of several genetic and biochemical changes in cells. Ultimately, these changes give cancer cells the ability to spread throughout the body or metastasize. Cancer cells display a variety of alterations to their cell surface carbohydrates. Cell surface glycoconjugates have been implicated in the adhesion, migration and invasion of cells, suggesting that changes to these structures may confer properties necessary for tumor cell metastasis. One such alteration is increased expression of β1-6 branched Asn-linked oligosaccharides on glycoproteins, which has been linked to the metastatic potential of cells. Hybridoma technology was used to generate monoclonal antibodies which detect glycoproteins bearing $\beta 1$-6 branched Asn-linked oligosaccharides which may be important in colon cancer. MAb 3A7 was selected for further study because it detected an epitope expressed at high levels in rat and human colon tumors. In addition, expression of the epitope defined by mAb 3A7 was shown to be developmentally-regulated in rat intestine. Thus, mAb 3A7 detected an oncodevelopmentally-regulated determinant in colon. As well, mAb 3A7 detects a major glycoprotein species of 140 kDa (gp140) which is differentially expressed in human colon cancer cell lines. MAb 3A7 recognizes an epitope containing blood group A (GalNAc$\alpha 1$-3Galβ-) or B (Gal$\alpha 1$-$3Gal\beta$-R) structures exclusively on type 2 chains (Gal$\beta 1$-4GlcNAc). 3A7-immunoreactive gp140 was isolated from the human colon cancer cell line, HT29, by lectin affinity and gel filtration chromatography. Partially purified gp140 was used to generate monoclonal antibodies which detect the polypeptide portion of gp140, namely mAbs 7A8, 7B11, 8C7 and 8H7. Immunological, molecular and biochemical analyses were used to demonstrate that the 3A7-immunoreactive gp140 corresponds to α3β1 integrin, a cell surface adhesion molecule which mediates cell-cell and cell-extracellular matrix interactions. Analysis of $\alpha 3\beta 1$ integrin expression in human colon carcinoma cell lines revealed that this glycoprotein is a major target for the addition of several cancer-associated carbohydrate structures, including $\beta 1$-6 branched Asn-linked oligosaccharides, poly-N-acetyllactosamine (type 2 chain repeats) and the 3A7 epitope. Significantly, the 3A7 epitope appears to be located primarily on the $\beta 1$-6 branch of Asn-linked oligosaccharides on $\alpha 3\beta 1$ integrin. Analysis of a panel of blood group A, AB and B positive human colon carcinoma cell lines revealed that expression of $\alpha 3$ integrin subunit, rather than glycosyltransferase levels, appears to regulate cell surface expression of the 3A7 epitope in colon cancer cell lines. Finally, $\alpha 3\beta 1$ integrin expressed by human colon cancer cells contributes to the adhesion and migration of cells toward extracellular matrix proteins. These data suggest that $\alpha 3\beta 1$ integrin and perhaps its glycan moiety, including the 3A7 epitope, contribute to colon cancer progression

Proteome analysis of metastatic colorectal cancer cells recognized by the lectin Helix pomatia agglutinin (HPA)

The lectin from Helix pomatia (HPA) binds to adenocarcinomas with a metastatic phenotype but the glycoconjugates of cancer cells that bind to the lectin have yet to be characterized in detail. We used a model of metastatic (HT29) and nonmetastatic (SW480) human colorectal cancer cells and a proteomic approach to identify HPA binding glycoproteins. Cell membrane proteins purified by HPA affinity chromatography, were separated by 2-DE and analyzed by MS. Competitive inhibition experiments with N-cetylgalactosamine, N-acetylglucosamine, and sialic acid confirmed that HPA binding was via a glycan-mediated interaction. Western blot analysis showed that HPA binds to proteins not recognized by an antibody against blood group A epitope. The proteomic study showed the main HPA binding partners include integrin αv/α6 and annexin A2/A4. These proteins were found complexed with microfilament proteins α and β tubulin, actin, and cytokeratins 8 and 18. HPA also bound to Hsp70, Hsp90, TRAP-1, and tumor rejection factor 1. This study revealed that the prognostic utility of HPA lies in its ability to bind simultaneously to many glycoproteins involved in cell migration and signaling, in addition, the proteins recognized by HPA are glycosylated with structures distinct from the blood group A epitope

Unprecedented diversity of genotypic revertants in lymphocytes of a patient with Wiskott-Aldrich syndrome

Spontaneous somatic reversions of inherited mutations are poorly understood phenomena that are thought to occur uncommonly in a variety of genetic disorders. When molecularly characterized, revertant cells have rarely exhibited more than one revertant genotype per patient. We analyzed individual allospecific T-cell clones derived from a Wiskott-Aldrich syndrome (WAS) patient identified by flow cytometry to have 10% to 15% revertant, WAS protein–expressing lymphocytes in his blood. Genotypic analysis of the clones revealed a remarkable diversity of deletions and base substitutions resulting in at least 34 different revertant genotypes that restored expression of WASp. A large fraction of these revertant genotypes were also identified in primary T cells purified from peripheral blood. These data suggest that the use of sensitive methods may reveal the presence of wide arrays of individual genotypic revertants in WAS patients and offer opportunities for further understanding of their occurrence