Vanessa I. Rasaiah

Efficient correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem/progenitor cells.

A deficiency in α-galactosidase A (α-gal A) activity causes Fabry disease. Virus-based delivery of genes can correct cells and establish a sustained supply of therapeutic proteins. Recombinant lentiviral vectors (LVs) show promise in this context. We first demonstrate LV-mediated marking of peripheral blood (PB) cells by transduction/transplantation of hematopoietic stem/progenitor cells. Stable enGFP expression was observed in PB for 37 weeks. Next, we transplanted Fabry mice with bone marrow mononuclear cells (BMMNCs) transduced a single time with a LV encoding the human α-gal A cDNA. Sustained expression of functional α-gal A in Fabry mice was observed over 24 weeks. Plasma α-gal A activity from treated Fabry mice was two-fold higher than wild-type controls. Increased α-gal A activity, often to supra-normal levels, and reduction of globotriaosylceramide, a glycolipid that accumulates in Fabry disease, was observed in all organs assessed. In secondary bone marrow transplantations, Fabry mice showed multilineage marking of PB, splenocytes and BMMNCs, along with therapeutic levels of α-gal A activity in plasma and organs over 20 weeks. Lastly, we transduced mobilized PB CD34+ cells from a Fabry patient and observed corresponding enzymatic increases. Thus a single LV-mediated transduction of primitive hematopoietic cells can result in sustained correction for Fabry disease.

Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2

Gene therapy strategies may accelerate the development of prophylactic immunotherapy against cancer. We synthesized a lentiviral (LV) vector encoding a kinase-deficient form of erbB2 (erbB2tr) to transduce murine dendritic cells (DCs) efficiently. Murine erbB2 models a clinically relevant tumor-associated self-antigen; its human homolog (HER-2/neu) is overexpressed in breast cancer and in 80% of metastatic prostate cancers. Following one infection, approximately 47% of DCs overexpressed erbB2tr. To determine whether low doses of transduced DCs could protect mice from prostate cancer cells, we performed prime/boost vaccinations with 2 x 10(3) or 2 x 10(5) erbB2tr-transduced DCs. Six weeks after vaccination, mice were simultaneously bilaterally challenged with the aggressive RM-1 prostate cancer cell line and an erbB2tr-expressing variant (RM-1-erbB2tr). Whereas control mice developed both tumors, all recipients of 2 x 10(5) erbB2tr-transduced DCs developed only wild-type RM-1 tumors. One-third of mice vaccinated with just 2 x 10(3) erbB2tr-transduced DCs also demonstrated erbB2tr-specific tumor protection. Protection against RM-1-erbB2tr tumors was associated with sustained levels of anti-erbB2tr antibody production and also correlated with erbB2tr-specific Th1 cytokine secretion. Depletion of CD4(+), CD8(+), or natural killer (NK) cells prior to tumor challenge underscored their role in mediating tumor protection. We conclude that administration of DCs expressing a self-antigen through efficient LV-based gene transfer activates cellular and humoral immunity, protecting host animals against specific tumor challenge.

Treatment of neutral glycosphingolipid lysosomal storage diseases via inhibition of the ABC drug transporter, MDR1 Cyclosporin A can lower serum and liver globotriaosyl ceramide levels in the Fabry mouse model

We have shown that the ABC transporter, multiple drug resistance protein 1 (MDR1, P‐glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, glycosphingolipid (GSL) synthesis. Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumulated glucosyl ceramide and Fabry cell lines of globotriaosyl ceramide (Gb3), by preventing de novo synthesis. In the Fabry mouse model, Gb3 is increased in the heart, liver, spleen, brain and kidney. The lack of renal glomerular Gb3 is retained, but the number of verotoxin 1 (VT1)‐staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice. Adult Fabry mice were treated with α‐galactosidase (enzyme‐replacement therapy, ERT) to eliminate serum Gb3 and lower Gb3 levels in some tissues. Serum Gb3 was monitored using a VT1 ELISA during a post‐ERT recovery phase ± biweekly intra peritoneal CsA. After 9 weeks, tissue Gb3 content and localization were determined using VT1/TLC overlay and histochemistry. Serum Gb3 recovered to lower levels after CsA treatment. Gb3 was undetected in wild‐type liver, and the levels of Gb3 (but not gangliosides) in Fabry mouse liver were significantly depleted by CsA treatment. VT1 liver histochemistry showed Gb3 accumulated in Kupffer cells, endothelial cell subsets within the central and portal vein and within the portal triad. Hepatic venule endothelial and Kupffer cell VT1 staining was considerably reduced by in vivo CsA treatment. We conclude that MDR1 inhibition warrants consideration as a novel adjunct treatment for neutral GSL storage diseases.

Direct injection of kit ligand-2 lentivirus improves cardiac repair and rescues mice post-myocardial infarction.

Myocardial infarction (MI) and subsequent adverse remodeling cause heart failure. Previously we demonstrated a role for Kit ligand (KL) in improving cardiac function post-MI. KL has two major isoforms; KL-1 is secreted whereas KL-2 is predominantly membrane bound. We demonstrate here first that KL-2-deficient mice have worse survival and an increased heart/bodyweight ratio post-MI compared to mice with reduced c-Kit receptor expression. Next we synthesized recombinant lentiviral vectors (LVs) that engineered functional expression of murine KL-1 and KL-2. For in vivo analyses, we directly injected these LVs into the left ventricle of membrane-bound KL-deficient Sl/Sl(d) or wild-type (WT) mice undergoing MI. Control LV/enGFP injection led to measurable reporter gene expression in hearts. Injection of LV/KL-2 attenuated adverse left ventricular remodeling and dramatically improved survival post-MI in both Sl/Sl(d) and WT mice (from 12 to 71% and 35 to 73%, respectively, versus controls). With regard toward beginning to understand the possible salutary mechanisms involved in this effect, differential staining patterns of Sca-1 and Ly49 on peripheral blood (PB) cells from therapeutically treated animals was found. Our data show that LV/KL-2 gene therapy is a promising treatment for MI.

Alpha-galactosidase A-Tat fusion enhances storage reduction in hearts and kidneys of Fabry mice.

The protein transduction domain from human immunodeficiency virus (HIV) Tat allows proteins to penetrate the cell membrane. Enhanced cellular uptake of therapeutic proteins could benefit a number of disorders. This is especially true for lysosomal storage disorders (LSDs) where enzyme replacement therapy (ERT) and gene therapy have been developed. We developed a novel recombinant lentiviral vector (LV) that engineers expression of α-galactosidase A (α-gal A)-Tat fusion protein for correction of Fabry disease, the second-most prevalent LSD with manifestations in the brain, kidney and heart. In vitro experiments confirmed mannose-6-phosphate independent uptake of the fusion factor. Next, concentrated therapeutic LV was injected into neonatal Fabry mice. Analysis of tissues at 26 wks demonstrated similar α-gal A enzyme activities but enhanced globotriaosylceramide (Gb3) reduction in hearts and kidneys compared with the α-gal A LV control. This strategy might advance not only gene therapy for Fabry disease and other LSDs, but also ERT, especially for cardiac Fabry disease.

Implementation of high-throughput screening for Fabry disease in Toronto dialysis patients

This bill is based on promotion of living-related donations, according to the ministry of health. A closely matched non-related person may only provide an organ out of compassion when a genetically-related donor is unavailable. Cadaveric donation is not among the designated priorities. The edict imposes strict penalties for brokers and those indulging in unsupervised removal, storage and grafting of organs. A federal monitoring authority and evaluation committees in designated institutions have been convened. A donor from their country of origin will have to accompany non-residents if they want to avail of our services [5]. Transplant registry, a nationwide pool of potential donors, a donor card system, as well as allocation of resources for disadvantaged end-stage renal disease (ESRD) patients, donor care and follow-up are planned. Transplant institutions will only be allowed to function if they (and personnel thereof) meet prescribed licensure criteria. Limited experience (28 cadaveric grafts to date), non-existence of a brain-death law and doubts about public approval of cadaveric mode of organ harvesting preclude the optimistic notion of the Transplant Society of Pakistan that a deceased organ donation programme will help save 50 000 lives every year. Pakistani law has global implications because of rampant transplant tourism [6]. It is consistent with contemporary trends [7]. Organ donation by relatives should be portrayed as an act of dignity. I suggest that a sustained strategy of directed donations be adopted, with more effective early identification of a genetically-related donor for each ESRD patient. A strong family setup, and motivation by specialist counsellors, may both play a critical role.