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Antisense RNA is a single stranded RNA, that acts complementary to the messenger RNA (mRNA) which is already transcribed within a cell. Similar to DNA, RNA also has an ability to form duplexes. 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. 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 (2). Stably integrated antisense genes which were used to inhibit specific genes were first demonstrated in bacteria (14) and in Eukaryotic cells (6). Genetic alteration of cells is carried out to express antisense RNA continuously which causes the target gene inhibition in the transduced cells. Genetic alteration of cells mediated via gene transfer, leads to the inhibition of virus replication, which is known as intracellular immunization (1). The use of antisense RNA technology is one method to obtain such inhibition.
ROLE OF ANTISENSE RNA IN MOLECULAR MEDICINE:
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:
HIV-1 antisense RNA transcripts with negative strand polarity were detected in the cell lines of the patients who were infected acutely and chronically with HIV-1 (19). One of these transcripts encodes HIV-1BRU open reading frame (189 amino acids) on the plus strand of genomic DNA, which is highly conserved among HIV-1 isolates (11). This antisense sequence is found to be complementary to the structured Rev- responsive element (RRE) and it extends through the cleavage site of Envelope protein (13). RRE, is present in all unspliced and singly spliced HIV transcripts. Malim et al., 1990 stated that binding of these RRE with Rev protein is necessary for the cytoplasmic expression of these transcripts. A study by Kem et al., 1996 detailed that expression of both sense and anti-sense RRE will inhibit the function of Rev. Thus from the results we can conclude that naturally occurring antisense RNA possessing antisense RRE, has the potential to reduce HIV gene expression and the accumulation of mRNA in liable cells.
The length of RNA duplex also plays a major role in the regulation of gene expression and long antisense RNA which is expressed in cells can inhibit the replication of heterologous strains of HIV-1 (approximately 50-80%) (15). The length of the duplexes formed between antisense RNA and primary RNA transcripts revealed a threshold value for their stability and efficiency. The length of an antisense ORF in HIV-1BRU is 570 bp. Nailya.E.Tagieva and Catherine Vaquero, 1997 reported that antisense HIV-1BRU RNA inhibits the replication of heterogenous HIV strains (III-B,NDK) in different cell types. The inhibition level was found to be 57% in transient transfected HeLaCD4 cells and 95% in cell lines which expressed antisense RNA constantly. Thus we can say that, the level of inhibition was diversified based on the cells.
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. Thus, it is evident that sub- region of HIV-1 against which the antisense transcripts are targeted plays a major role in the level of inhibition of HIV-1 replication (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. Secondary structures which are assumed to affect the inhibitory properties are thought to be present on long antisense RNA transcripts are inapplicable when compared with short oligonucleotides (16).
Suitable Vector Construction Enhances Inhibition:
Generally, Retroviral vectors, are used for introduction of transgenes, gene Â therapeutic antiviral agents and expression of antisense or sense RNA in various cells. These vectors are known as effective gene transfer vehicles, since they have the capability of integrating into target cell's genome. In this case it is the antisense RNA that is to be carried with the vector. The effectiveness of anti-HIV gene therapy through retroviral vectors has been tried out in different T- cell lines and in primary CD4+ T- lymphocytes (8). Since we know that, the major targets for HIV infection are primary cells; there arises limitations in this strategy (12). To overcome this limitation, transduced PBLs were used to test the potency of antiviral constructs to inhibit HIV 1 expression in these cells. The results based on above methodology revealed that retroviral vector- mediated expression of an antisense HIV-BRU RNA causes inhibition of heterologous strain 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 natural antisense HIV-1 sequence is sustained among different HIV-1 isolates and is found to be expressed in various types of cells (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 and accumulation of mRNA in susceptible cells. As soon as RNA duplexes are formed they act as targets for nucleases which degrade the dsRNA region. This leads to decrease in HIV mRNA accumulation. As a result, antisense transcript is also reduced in the transduced cells. (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.