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RNAi against HIV infection
#1
Study of RNAi based therapy for HIV:

The trigger of RNA interference in response to the double stranded RNA has become one of the vastly studied areas of molecular biology and this RNA interference effect has become a genetic tool in the gene function studies as well as the development of therapeutics for various diseases by manipulating genes and their related functions. The control of the gene function helps in the regulation of the different developmental stages in the life cycle of an organism as well as in the progression of the various stages of a particular disease.

The first target of the RNAi was the infectious agent responsible for HIV infection known as the Human Immunodeficiency virus. The reason behind this development maybe due to extensive research in this area leading to accumulation of knowledge regarding the life cycle of the virus and its pattern of gene expression. Further research has proved the role of RNAi in targeting other diseases such as Hepatitis B, Hepatitis C, Polio, etc.

Several HIV-encoded RNAs both early and late have been targeted in cell lines as well as in the primary haematopoietic cells including the tat, gag, pol, env, nif, rev, vpr, TAR element, reverse transcriptase, Long terminal repeat (LTR), etc by the expressed shRNAs (short hairpin RNAs) and the siRNAs prepared synthetically. In certain cases, RNAi has been illustrated in the prevention of HIV infection in cells i.e. before they are infected by the retrovirus. However, in cases where the cells are infected by the HIV retrovirus, the RNAi mechanism proceeds via the following steps:
a) The release of the RNA genome of the retrovirus transcribes with the help of HIV reverse transcriptase to form HIV-DNA, also known as the provirus that incorporates into the cell genome and gives rise to mRNA transcripts.
b) Artificially synthesised siRNA specific to the HIV mRNA in a particular stage of the virus life cycle are introduced into the cell by injection or lentivirus vectors called siRNA vectors.
c) These siRNAs inserts into the RISC (RNA-Induced Silencing complex) whereby only a single strand of siRNA remains, that binds to the specific HIV mRNA and cleaves it. RNases within the cell then remove these fragments.

Thus, the siRNAs help in the neutralization of the HIV mRNAs thereby reducing the chance of synthesis of HIV proteins within the cell thus preventing the progression of the infection.

Although, the inhibition of HIV-encoded RNAs mediated by RNAi has been made possible, the direct targeting of the HIV virus faces numerous challenges for clinical application due to an increased rate of mutation in the virus leading to the formation of mutants that escape from being targeted. This incidence of mutation is observed in not only HIV viral RNAs but also other RNA viruses encoding RNA Polymerases or reverse transcriptase, which also have a tendency of formation of mutants with every replication cycle. Hence, complementary approach of targeting of cellular transcripts that encode for functions such as the entry and replication of HIV virus i.e. the down-regulation of the various cofactors present within the cell necessary for the progress of HIV infection was studied.


Down-regulation of various cellular cofactors such as the HIV receptor CD4, NFκB, and the co-receptors CCR5 and CXCR4 have been studied, which proved successful in the replication of the virus as well as its cell entry. However, the CXCR4 receptor was found to be essential for the hematopoietic stem cell formation in the bone marrow as well as subsequent T cell differentiation and the CD4 was also found to be an essential cellular receptor. Hence, although the targeting of these cofactors was found to help in complete stoppage of viral replication, it created several problems within the body. Hence, viral targets became essential for the successful study of RNAi based therapy for HIV. The mixture of single shRNAs with several antiviral genes have become a potent alternative anti-HIV approach, which is becoming an active area of research.


The introduction of the siRNAs have posed great challenge as the vectors used for the delivery initiated immunological reactions within the body. It was overcome partly by an approach involving the isolation of the T-cells of patients, its transduction with lentiviral vector carrying the anti-HIV antisense RNAs and expansion, followed by re-infusion into the patient’s blood stream. Another significant progress in this area is the use of the hematopoietic progenitor stem cells, which are isolated, transduced with the vector carrying the therapeutic genes, followed by reinfusion.


In this way, it can be noted that compared to the ribozymes or the antisense approaches, the RNAi based therapy is more potent. The pre-clinical study of this approach on the human trials in near future will mark a revolution in the therapeutics for the HIV based infection.
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#2
HIV and RNA interference

The original article in this thread points out some of the challenges associated with adopting an RNA interference-based approach to HIV therapy. These challenges include the high mutation rate of the virus and difficulties inherent in targeting therapeutic agents to the desired site of action.

The high mutation rate of the virus supports the notion of targeting multiple genes of the virus. This also has the advantage of allowing gene expression to be targeted at critical time points in the viral replication pathway. A group from the Beckman Research Institute in Duarte in the USA examined the potential of intronic MCM7 (minichromosome maintenance complex component-7) platform as a way of expressing small inhibitory RNAs against HIV. This platform normally harbours endogenous microRNAa (miRNAs) and these were replaced with multiple anti-HIV small RNAs targeting genes including tat and rev. The study found that three of the combinatorial constructs tested were able to potently suppress viral replication in HIV-1-infected CEM T lymphocytes when compared with cells that had not been transduced with a construct. One of the most effective constructs combined anti-HIV siRNA with a nucleolar- targeting U5 ribozyme and a trans-activation response (TAR) decoy designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. The group maintained that their constructs represent ‘a new paradigm for combinatorial RNA-based gene therapy applications’.

Another study from the same institute addressed the issue of which small interfering RNA strand is chosen by the RNA-induced silencing complex (RISC) for gene silencing and how it is targeted. It is important that the intended strand is selected and that the unintended strand is blocked to prevent off-target effects and increase potency of the silencing effect. The group used the technique of unlocked nucleic acid (UNA) modification of the 5' end of canonical siRNA, which abrogates gene silencing of the modified strand and demonstrated that modifying the unintended strand in their HIV siRNA resulted in improved targeting by the intended antisense strand in otherwise poorly targeting siRNA.

Much more work is needed to continue to develop RNA interference in HIV therapy but the signs are promising.

Sources

CHUNG, J. et al., 2012. Endogenous MCM7 microRNA cluster as a novel platform to multiplex small interfering and nucleolar RNAs for combinational HIV-1 gene therapy. Human Gene Therapy, 23(11), pp. 1200-1208

SNEAD, N.M. et al., 2013. 5' Unlocked Nucleic Acid Modification Improves siRNA Targeting. Molecular Therapy.Nucleic Acids, 2, pp. e103-e103
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