Tuberculosis

Projects

Nanocos: chitosan oligosaccharide-siRNA nanoplexes for inhibiting intracellular Mycobacteria (INVITED)

Principal Investigator Dr. Prajakta Dandekar, UICT

Collaborator The University Institute of Chemical Technology (UICT), Mumbai, India

Co-investigator Dr. Nerges Mistry, FMR

Project team Dr. Rahul Upadhyay, Dr. Purva Bhatter

Funder Department of Biotechnology, Govt. of India

Duration June 2013- May 2015

Budget Rs.7.92 Lakhs

Background

M. tuberculosis (M.tb) is an intracellular parasite acquiring resistance to the existing drugs. The intracellular bacterial lifestyle allows it to be “hidden” from the immune system and thus divide and cause disease. It is well known that M.tb interferes with the programmed cell death pathway of its natural host (macrophages) thereby actively dividing in the host cell. While most drugs target bacterial mechanisms to be blocked, the use of mechanisms that causes disequilibrium of host-pathogen relationship is less explored.

The advent of new technology such as silencing of genes through siRNA based technique allows repressing the expression of specific genes thereby allowing assessment of its impact on specific cellular functions.

The current study undertaken in collaboration with UICT will allow assessing the potential of siRNA particles, targeted towards bfl-1 gene, in controlling the growth of Mtb. bfl-1, is a part of the caspase 2 cluster involved with programmed cell death regulation. Expression of bfl-1 opposed/blocks apoptosis and allows proliferation of M.tb. The use of siRNA targeting aims to block bfl-1 expression and thus allow apoptosis to occur which would eventually result in clearance of M.tb proliferating intracellularly.

Objective

To study the effect of Nanocosbfl1 siRNA on the intracellular growth of M.tb

Role of UICT

Production of siRNA and its characterization

Role of FMR

  • Testing the specificity of siRNA, through use of quantitative real time PCR: checking for the expression of bfl-1 post treatment with the generated siRNA’s,
  • Determining the cidal potential of the siRNA particles through use of routinely practiced in-vitro macrophage model,

Technology Transfer

FMR has requested UICT to train one of its personnel for preparation, assembly and characterization of siRNA particles.

Progress of work

Expression analysis of bfl-1 and gapdh in the presence of targeted siRNA particles along with scrambled siRNA as control with Pre and Post-infection models with H37Rv in mouse peritoneal macrophage using Real-time PCR.

Pre-infection model—contains peritoneal macrophage + Nanoparticle treatment and then M.tb infection is established.

Post-infection model— contains peritoneal macrophage + M.tb infection and later Nanoparticle treatment

Positive control (transfection reagent + Nanoparticle siRNA, naked siRNA) and negative controls (scrambled siRNA in nanoparticle, no particle and no infection)

Key findings

Nanoparticle along with bfl-1 siRNA in both pre-infection and post-infection model showed no expression of bfl-1 gene demonstrated using Real-time PCR indicating complete silencing of the gene. The siRNA with nanoparticle silence the bfl-1 expression irrespective of the use of the transfection agent. The Naked siRNA particles both the target and the scrambled do not block the gene expression of either bfl-1 orgapdh. It is likely that the naked siRNA particles are not trans-located across the cell membrane or are denatured in the lysosomal compartment. The gapdh expression was unaltered, indicating the efficacy of the siRNA particle. However, the siRNA (scrambled) particles with and without the transfection agent also silence the bfl-1 expression leaving the gapdh expression unaltered. This could be due to non-specific binding of the scrambled particles to the bfl-1 gene. Similarly, the transfection agent also silences the expression of bfl-1.The results of negative and positive controls used indicate the specificity and sensitivity of the technique.

A recent experiment demonstrated that silencing of bfl-1 gene occurs using targeted nanoparticle delivery of siRNA which in-turn indicates induction of apoptosis pathway after infection and eventually clearing of the infection.

Plan of Work

The scrambled siRNA needs to be tested for its specificity as it showed non-specific silencing of bfl-1gene in the experiment. Therefore, the experiment has to be performed to study the silencing of bfl-1 gene using nanoparticle along-with siRNA and unrelated scrambled siRNA control.

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