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.
(565 words)
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