Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing beta-galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175-200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.
Upregulation of heat shock proteins (HSPs) is an approach to treatment of neurodegenerative disorders with impaired proteostasis. Many neurons, including motor neurons affected in amyotrophic lateral sclerosis (ALS), are relatively resistant to stress-induced upregulation of HSPs. This study demonstrated that histone deacetylase (HDAC) inhibitors enable the heat shock response in cultured spinal motor neurons, in a stress-dependent manner, and can improve the efficacy of HSP-inducing drugs in murine spinal cord cultures subjected to thermal or proteotoxic stress. The effect of particular HDAC inhibitors differed with the stress paradigm. The HDAC6 (class IIb) inhibitor, tubastatin A, acted as a co-inducer of Hsp70 (HSPA1A) expression with heat shock, but not with proteotoxic stress induced by expression of mutant SOD1 linked to familial ALS. Certain HDAC class I inhibitors (the pan inhibitor, SAHA, or the HDAC1/3 inhibitor, RGFP109) were HSP co-inducers comparable to the hydroxyamine arimoclomol in response to proteotoxic stress, but not thermal stress. Regardless, stress-induced Hsp70 expression could be enhanced by combining an HDAC inhibitor with either arimoclomol or with an HSP90 inhibitor that constitutively induced HSPs. HDAC inhibition failed to induce Hsp70 in motor neurons expressing ALS-linked mutant FUS, in which the heat shock response was suppressed; yet SAHA, RGFP109, and arimoclomol did reduce loss of nuclear FUS, a disease hallmark, and HDAC inhibition rescued the DNA repair response in iPSC-derived motor neurons carrying the FUS P525L mutation, pointing to multiple mechanisms of neuroprotection by both HDAC inhibiting drugs and arimoclomol.
Expression of the coxsackie and adenovirus receptor (CAR) is downregulated in malignant glioma cell lines and is barely detectable in high-grade primary astrocytoma (glioblastoma multiforme). We determined the effect of forced CAR expression on the invasion and growth of the human glioma cell line U87-MG, which does not express any CAR. Although retrovirally mediated expression of full-length CAR in U87-MG cells did not affect monolayer growth in vitro, it did reduce glioma cell invasion in a 3-dimensional spheroid model. Furthermore, in xenograft experiments, intracerebral implantation of glioma cells expressing full-length CAR resulted in tumors with a significantly reduced volume compared to tumors generated by control vector-transduced U87-MG cells. In contrast, U87-MG cells expressing transmembrane CAR with a deletion of the entire cytoplasmic domain (except for the first 2 intracellular juxtamembrane cysteine amino acids) had rates of invasion and tumor growth that were similar to those of the control cells. This difference in behavior between the 2 forms of CAR was not due to improper cell surface localization of the cytoplasmically deleted CAR as determined by comparable immunostaining of unpermeabilized cells, equivalent adenoviral transduction of the cells and similar extent of fractionation into lipidrich domains. Taken together, these results suggest that the decrease or loss of CAR expression in malignant glioma may confer a selective advantage in growth and invasion to these tumors.
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