Overexpression of Human Methylmalonyl CoA Mutase in Mice afterIn VivoGene Transfer with Asialoglycoprotein/Polylysine/DNA Complexes

Abstract

Methylmalonic acidemia resulting from genetic deficiency of methylmalonyl CoA mutase (MCM) is an often fatal metabolic disease. Somatic gene therapy for this disorder may require gene replacement in the liver. We describe overexpression of MCM in the liver of mice after in vivo gene delivery using asialoglycoprotein/polylysine/DNA (ASO/PL/DNA) targeted delivery to the liver of plasmids expressing recombinant MCM. After intravenous administration of the ASO/PL/DNA complex, the vector sequences are cleared from the blood with t1/2 = 2.5 min and > 95% of the vector is taken up by the liver. Vector sequences are cleared from the liver with t1/2 = 1.0-1.3 hr. MCM enzyme activity in the liver increases to levels 30-40% over baseline 6-24 hr after injection. No acute or chronic toxicity was observed. This net level of expression is likely to be therapeutic for MCM if the complex could be administered repetitively to treat acute episodes of life-threatening acidosis or establish a steady-state level of MCM activity. Repetitive administration of the ASO/PL/DNA complexes in mice was associated with formation of antibodies against asialo-orosomucoid and the asialo-orosomucoid complex but not against DNA.

Living Donor Liver Transplantation for Pediatric Patients with Inheritable Metabolic Disorders

“…However, all of these reported therapies were symptomatic treatments and the evidence of their efficacy seemed to be anecdotal. To achieve a satisfactory outcome in the treatment for NLOD, a breakthrough of some sort will be needed, such as development of a gene therapy (24)(25)(26) to eradicate the intrinsic underlying disorders. At this time, however, LT combined with these reported symptomatic therapies is the sole therapeutic procedure for NLOD patients with severe manifestations.…”

3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins

“…Directing nonviral vectors to cellular receptors by the incorporation of specific ligands, such as transferrin, asialoglycoprotein, or epithelial growth factor (EGF), has provided an efficient means of crossing the plasma membrane by receptor-mediated internalization. [1][2][3][4][5][6][7][8][9][10][11][12][13] The cellular endosome encapsulating the vector following endocytosis, however, has proven to be a major barrier to nuclear delivery and gene expression by nonviral vectors. 14 Without endosome penetration, lysosomal enzymes eventually degrade the DNA, resulting in low transduction.…”

Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo

“…[24][25][26][27][28][29][30] However, the in vivo efficiency of such targeted vectors is more rarely documented. 31,32 For cationic lipids and PEI, transfecting capacity might require either an intrinsic capacity to destabilize the endosomes or the association with a helper lipid such as DOPE, [33][34][35][36] and has been shown to be enhanced by the addition of amphiphilic peptides. 37 Some attempts were made to evaluate the potential of combining DNA-compacting agents such as poly-l-lysine or histones with cationic lipids, describing some enhancement of transfection performed in serum-free in vitro conditions, 13,21,[38][39][40][41][42][43] one article reported increased transfection in the presence of serum, 13 and one interesting paper described the use of cationic-lipid-protamine sulfate-DNA complexes for in vivo gene transfer by intravenous administration.…”

“…Additionally, the use of large proteins or polypeptides, either natural or chimeric, would increase the risk of inducing an immune response, either against the protein itself or against the transfection complex as already described for the poly-l-lysine/asialo-orosomucoid/ DNA complexes. 32 An alternative approach to proteins or cationic polymers for combining to cationic lipids, could be the use of a peptide -as short as possible and preferably of natural sequence. This would allow chemical synthesis, thereby reducing the production and purification difficulties.…”