scientific article -Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice

Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice

Introduction

This article talks about a lysosomal storage disorder that is caused by the deficiency of the lysosomal enzyme arylsulfatase A(arsa),that is coupled with neurological impairment. This disease is characterized by myelin degeneration in the CNS and PNS. Due to the accumulation of ARSA substrate called galactosylceramide I3-sulfate.

This diseases can only be diagnoised in humans after the onset of symptoms so in order to solve the neurological disease a new strategy had to be devised. Hence through this article they have proven the complete normalization of established behavorial abnormalities,motor conduction and neruopathological conditions of ARSA(-/-) mice upon HSPC-GT(GENE-TRANSFER)

In this research they made use of macrophages and microglia cells to release the desired lysosomal enzyme so that in-vivo cross correction can occur

The animal model they made use for this research was a mouse model of MLD which was characterized with slow progressive CNS and PNS disease. They display delayed motor conduction,neuronal degeneration and widespread storage of metachromatic material that further amplifies along with age.

The experiment was kicked off by transplanting HSPC’S from ARSA donors using a vector(LV) THAT WAS EXPRESSIN THE ARSA gene. To optimize the detection of this enzyme in the mouse they made use of a C-terminal-tagged transgene that contains the HA peptide from the human influenza virus.

The mouses were also further divided into group A and group B due to the difference in the average ARSA ACTIVITY which were lower in the first 4 transplantation group(group A) than compared to the last 4 which was higher.(group B).

So how did this research manage to correct the neurological problem ?- through the transplantation of the HSPC’S and the GT to over express the lysosomal enzyme in the brain using the microglia  the deficits were managed to be normalized, and there was the prevention of demyelination even surprisingly the remyelination in the PNS which in turn might account for reversal of motor conduction defects.

The methods used for this experiment were motor learning tasks that were performed with an accelerating rotarad apparatus so as to mesure the performance of the mouses. Histopathology for eg semithin and ultrathin morphological analyses were performed. Transduction of hemopoietic progenitors and HSCT were performed by using the stem separation kit first then the vector was injected via the tail vein into the mouse.

The ARSA assays were performed by plating BM cells in a methylcellulose-based medium. After 10 days colonies were plucked and lysed for PCR analysis and for the detection of LV sequences.  immunofluorescence analysis and western blot analysis was carried out and finally for ARSA-HA specific activity detection a p-NC assay was adapted.

 Finally there were safety concerns of using LV gene transfer and the enzyme overexpression  in HSPC’S, this is because there is a NEED  for an high engraftment of transduced cells that relies in multiple vector integration which can lead to integration-dependant genotoxicity and there was also the  requirement of supranormal levels of enzymes to be produced so as to achieve significant therapeutic effects in the nervous system. However this problem was addressed by making use of the LV vector that compared to using the retrovector (RV)which suggests the risk of insertional mutagenesis could be significantly lower that compared to using RV’S, and hence through the use of LV the researches never detected any hematopoietic abnormalities.

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