RNA has an ability to form duplexes, similar to DNA. It needs a second strand of RNA, which has its sequences exactly complementary with respect to the first strand. The second strand is known as the antisense strand, since it has nucleotide sequences complementary to message sense. This process is known as "direct inhibition". Thus when, mRNA forms a duplex with complementary antisense RNA sequence, translation is blocked as a result. In the case of "indirect inhibition", the sense sequences interact with the binding proteins or with the enzymes involved in the metabolism of nucleic acids. These interactions need not be gene specific (23). This is because either the ribosome cannot enter the nucleotides in mRNA or the ribonucleases degrade the duplex RNA rapidly. "Non-specific inhibition" also occurs when there is an increase in concentration of antisense oligonucleotides (22). The Antisense RNA are now in use for anti-HIV gene therapy. It specifically inhibits expression of few genes by stably integrating with them. This was shown in bacteria (14) and in Eukaryotic cells (6). The method of using antisense RNA by gene transfer in to cells which results to the inhibition of viral replication is called intracellular immunization (1).
ROLE OF ANTISENSE RNA IN MOLECULAR MEDICINE:
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Nucleic acids can be used as a curative medicine since they add up to the basis of antisense and gene therapies. One of the major applications of the antisense strategy is to regulate the transcription of disease-related genes in vivo. They have the ability to identify their target in a highly sequence-specific manner and thus they are useful in inhibition of unusual gene expression and pathogenic viral functions. Cells infected with human immunodeficiency virus type -1 (HIV-1) or different forms of leukemia have enormously enhanced the prospects of antisense research (4).
INHIBITION OF HIV TYPE-1 BY ANTISENSE RNA EXPRESSION:
Various studies have been conducted to determine the role of Antisense RNA in inhibiting the HIV type -1 replication. From these studies, naturally occurring antisense RNA have been identified. This knowledge has been applied to the development of artificial antisense RNA.
NATURALLY OCCURING ANTISENSE RNA INHIBITS HIV TYPE-1 REPLICATION:
Nailya E.Tagieva analyzed HIV-1BRU ORF on the plus strand of genomic DNA. This ORF is encoded by one of the HIV-1 antisense transcripts detected in the cell lines of the patients who were infected acutely and chronically with HIV-1 (19). A segment of length 189 nucleotides in this antisense ORF sequence is complementary to the rev response element (RRE) region found in all unspliced and singly spliced HIV RNA transcripts (13). Rev response element codes for rev protein which directs the export of RNA transcripts from nucleus to cytoplasm for their expression (9). So binding of antisense ORF sequence to this RRE region inhibit the viral gene expression.
Due to these reasons a study was carried out by Nailya E.Tagieva to analyze the above inhibition in presence of over expressing natural antisense- RNA containing ORF. Formation of RNA-RNA hybrid involves structural constraints. Stable Secondary structures in complementary region prevent binding of antisense-RNA containing ORF to RRE (15). This hybrid formation requires helper proteins and structural features to enhance the interaction between them (24). Thus from the results we can conclude that naturally occurring antisense RNA possessing antisense RRE, has the potential to reduce HIV gene expression.
HIV-1 targeted antisense RNA is found constantly in free, non- injected cells. This may drive to "intracellular immunity" against successive HIV-1 infection. Thus the application of antisense RNA by intracellular expression has been used (16). Karola Rittner and Georg Sezatiel, 1991 conducted a study, in which fivefold molarplasmids expressing antisense RNA regions which are exactly complementary to set of HIV-1 target regions was co-microinjected into the proviral HIV-1 DNA. This is done to test their antiviral activity (16). Approximately 75% inhibition was observed when antisense RNA sequence is complementary to the coding sequences of HIV-1 regulatory proteins (7).
Antisense oligodeoxyribonucleotides which are modified chemically are also used for the inhibition of HIV-1 virus replication (21). They are added to the culture medium. It was shown that antisense oligonucleotides which are added complementary to LTR sequences and splice sites inside the HIV-1 genome, act as virus inhibitors (4). But there also occurs some disadvantages, because cellular uptake and stability within the cell cannot be controlled.
Suitable Vector Construction Enhances Inhibition:
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Generally, Retroviral vectors are most commonly used as vehicle for efficient gene transfer since it gets integrated with genome of target cells (12). Retroviral vectors have been used for transfer of functional HIV proteins and its efficiency has been tested in different cell lines and in primary CD4 lymphocytes (15). In this case it is the antisense RNA that is to be carried with the vector. Since we know that, the major targets for HIV infection are primary cells; there arises limitations in this strategy. To overcome this limitation, a study was carried out on different cell lines transduced with antisense RNA carrying retroviral vectors (12).The cell lines transduced with vectors carrying mutated antisense RNA insert or lacking insert as control were used (12). The results reveals that this causes inhibition of HIV-1 replication in primary T-lymphocytes.
The efficiency of a vector in transporting the antisense RNA and producing the desired effect depends on the way it is constructed (5). From this we see that there are two important considerations for an "efficient vector".
Vector copy (single or double)
Various promoters are used in vectors, like MLV, SV40, HIV, etc. Choosing the right promoter would increase the gene expression. While considering the promoters considered as suitable candidates for HIV inhibition, tRNAmet human promoter has been found to be of high efficiency than its counterparts (5). In-vitro studies show that, compared to single copy vectors, double copy vectors are highly efficient in inhibiting the replication of the HIV by interacting with their nucleotides (20).
Antisense RNA acts as down regulators of various steps involved in the gene expression. This leads to a reduction or prevention of host cell infection (18). We can say that antisense RNA are highly- specific virus inhibitors. The naturally occurring antisense HIV-1 sequence is found to be expressed in various types of cells in all the individuals affected with HIV-1 (11). In the meantime, a negative-strand HIV-1 promoter has also been identified (10).This will help in enhancing the knowledge of design of antisense RNAs.
Naturally occurring antisense RNA with the presence of antisense RRE has the ability to inhibit HIV gene expression (12). Designing a suitable vector is of prime importance for an efficient inhibition. Double copy vectors seem to be a good candidate for transferring antisense RNA (20). Antisense RNA is likely to have a promising future in the treatment of other type of diseases.