Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype

Abstract

IntroductionGlycogenosis type II (Pompe disease, acid maltase deficiency; Online Mendelian Inheritance in Man no. 232300) is an autosomal recessive lysosomal storage disorder caused by acid ␣-glucosidase (GAA) deficiency. The disease is characterized by glycogen storage in liver, spleen, kidney, brain, and endothelial cells and most prominently in skeletal, heart, and smooth muscles. Symptoms arise from muscular weakness and wasting. Infants with complete enzyme deficiency present shortly after birth, lose all muscle strength within 8 months, and succumb to hypertrophic cardiomyopathy and respiratory failure in the first year of life. 1 Children and adults with residual GAA activity show a more protracted course and may become wheelchair bound, dependent on artificial ventilation, and have a shortened life expectancy. Presently, enzyme replacement therapy (ERT) based on intravenous infusion of recombinant human ␣-glucosidase, taken up by mannose 6-phosphate receptor mediated endocytosis, 2,3 is a major therapeutic advance that prolongs the life of affected infants but does not guarantee long-term symptom-free survival, requires biweekly administration, and may induce immune responses to the recombinant protein.As an alternative to ERT, in vivo gene therapy mediated by adenoviral vectors and adeno-associated virus vectors (AAVs) has been investigated in a mouse model of Pompe disease. [4][5][6] However, long-term efficacy can be significantly hampered by antibody formation, 7,8 and adverse immune responses to the vector has been observed after adenoviral and AAV gene therapy in patients. 9,10 For treatment of patients with other lysosomal enzyme deficiencies, allogeneic hematopoietic stem cell (HSC) transplantation has been proposed. 11 HSC transplantation proved effective in ameliorating the neurologic symptoms in murine globoid cell leukodystrophy and human patients 12,13 as well as in mucopolysaccharidosis I (Hurler syndrome). 14,15 Other lysosomal storage disorders such as metachromatic leukodystrophy (MLD) may require higher enzyme levels than provided by HSC transplantation; lentiviral (LV) vector-mediated overexpression of aryl-sulfatase A in HSCs effectively reversed the neuropathologic phenotype in the mouse model. 16 In addition, LV-mediated clinical gene therapy in trial phase of X-linked adrenoleukodystrophy halted progressive cerebral demyelination in 2 patients. 17 Recently, HSC transplantation was shown to promote immune tolerance to ERT in the Pompe mouse model. 18 The use of gene-modified autologous HSCs also overcomes the profound conditioning and immune barriers associated with allogeneic transplantation.The few attempts of HSC transplantation for Pompe disease have not met with success. 19 GAA levels, if any, are low in hematopoietic cells in mice, 18 and allogeneic transplantation is not an obvious treatment. Therefore, high-level vector-driven ectopic expression of the enzyme in hematopoietic cells would be required to accomplish efficacy. We tested the hypothesis that ex vivo LV For persona...

Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells

“…bPRE4*.SIN (LV.eGFP) containing the spleen focus-forming virus promoter or the phosphoglycerate kinase (PGK) promoter (LV-PGK-eGFP) was produced as described previously. 16 The vector backbone was kindly provided by Dr Naldini with modifications by Dr Schambach. 16 Lentivirus vector stock physical titers ranged between 1.2 ϫ 10 9 and 9 ϫ 10 12 p24 vector particles (VP/mL) determined by QuickTiter Lentivirus Quantitation Kit (Cell Biolabs, Inc) for the detection and quantitation of lentivirus-associated HIV-1 p24 core protein.…”

“…16 Lentivirus vector stock physical titers ranged between 1.2 ϫ 10 9 and 9 ϫ 10 12 p24 vector particles (VP/mL) determined by QuickTiter Lentivirus Quantitation Kit (Cell Biolabs, Inc) for the detection and quantitation of lentivirus-associated HIV-1 p24 core protein. Biologic titers ranged between 1.7 ϫ 10 6 and 1.9 ϫ 10 9 transducing units (TU/mL), determined on CMS5 31 cells or HeLa cells as described previously, 16,32 and are referred to as applied multiplicity of infection (MOI).For lentiviral magselectofection of human umbilical cord blood mesenchymal-like stem cells (hUC-MSCs) and hCB-CD34 ϩ cells, virus vector was mixed with SO-Mag2, resulting in a composition up to 20 fg Fe/VP and kept at room temperature for 20 minutes to allow complex assembly. The characteristics of the complexes are summarized in supplemental Table 3.…”

Infantile pompe disease presenting itself with severe hypertrophic cardiomyopathy: three case reports

“…Gelecekte ki tedavi hedefi gen tedavisidir (10). Ülkemiz koşullarında indeks vaka durumunda neonatal dönemde tanı amaçlı erken dönemde kan alınıp ERT başlanmasının kardiyak bulgularda düzelme ve prognoza iyi yönde etki edebileceğini düşünüyoruz.…”

New insights into therapeutic options for Pompe disease

“…Autologous HSC gene therapy with integrative viral vectors would improve the strategy by alleviating the GVHD and allowing high-level GAA expression/secretion from strong promoters. The efficiency of HSC-gene therapy was demonstrated in GAA-KO mice after transduction of HSC with lentiviral vectors (56,57). The overexpression of hGAA in hematopoietic cells resulted in a major clearance of glycogen in heart, diaphragm, and liver leading to cardiac remodeling, restoration of respiratory function, as well as muscular strength improvement.…”

“…The induction of immune tolerance to the therapeutic enzyme would greatly enhance the benefit of ERT in these patients (58), but no immune modulation or tolerization protocol has permitted to maintain the efficacy of ERT after the formation of anti-GAA antibodies. Whereas ubiquitous expression of therapeutic proteins provoked antibody formation and cytotoxic T lymphocyte (CTL)-mediated response, liver-specific promoters induced immune tolerance to therapeutic proteins in immune competent GAA-KO mice (52)(53)(54)58 (56,57). Immunomodulatory agents already used in clinical practice such as mycophenolate (MMF), methotrexate (MTX), and cyclosporine A/azathioprine (CsA/Aza) were evaluated in GAA-KO mice (60).…”