In the past 10 years many molecular methods have been developed to cure different diseases. Molecular methods can be used to identify common viral pathogens not easily detectable by culture or antigen tests ex: - hepatitis C virus tests (HCV) and HIV. Such methods are becoming rapid and inexpensive enough to replace less sensitive antigen tests. Molecular biology will have a profound impact upon the treatment of diseases. Molecular techniques provide treatment of more diseases each year. The understanding of patho-physiology allows for improved drug design. Antisense technology can relatively control gene expression. Gene therapy is potentially the most imp aspect of molecular biology. Physical and viral transduction mechanisms are being developed towards this end. Gene replacement, creation of antisense oligo-nucleotides and prodrug strategies are being developed.
Methods of treatment for diseases at molecular level;
There are many methods of treatment of diseases at molecular level. Out of those the three that are extensively used are discussed below. They are
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Gene replacement therapy
Gene replacement therapy:
Gene therapy is the use of genetic material (DNA) inserted into patients cell for the treatment of inherited or acquired diseases. They are many medical conditions that are result of mutations in patients gene. Gene therapy tries to introduce functional form of that gene into the patients cell nucleus in order to compensate for the mutated gene.
Antisense is a form of treatment for growth disorder of infections. When the genetic sequence of a particular gene is known be causative of a particular disease. It is possible to synthesize a strand of nucleic acid (DNA, RNA or chemical analogue) that will bond to messenger RNA(mRNA) produced by that gene and inactivate it, effectively turning that gene off. This is because mRNA has be single strand might be targeted to bind a splicing site on pre-mRNA and modify the exam content of an mRNA. 
This synthesized nucleic acid is termed an "anti-sense" oligo nucleoside because its base sequence is complementary to the gene's messenger RNA (mRNA), which is called "sense" sequence so that a sense segment of mRNA "5'-AAGGUC - 3' " would be blocked by the antisense mRNA segment "3'- UUCCAG - 5' ".
Many cancers including lung cancer, colorectal carcinoma, pancreatic carcinoma, malignant glioma and malignant melanoma, and other diseases such as asthma, arthritis, diabetes, muscular dystrophy and ALS are researched to treat by antisense drugs.. The identification of appropriate protein target is most important for the use of antisense strategy. This approach is very easy to use as it allows the use of oligonucleotides against any over expressed protein so that the mRNA that codes for that protein is known..
Antisense therapeutic approaches can be applied to several classes of molecular targets in diseases.
As in viral infections, proteins are expressed from foreign or exogenous genes. The major immediate early region2 of hCMN inhibition of which prevents viral replication.
As a part of disease process proteins are over expressed or up regulated. One of the example is the antisense targets are cytokines or other inflammatory mediators such as ICAM-1.
In the rate-limiting steps of the disease process, proteins are active. Here the proteins need not necessarily be over expressed to be disease target. Ex: antisense disease target include cytokine receptors essential for cell division, such as the insulin like growth factor (IGF) - I receptor in epidermal keratinocytes..
In future this antisense technique will definitely make its impact on biotechnology and medicine.
RNA interference therapy is an approach of preventing some of the cells messenger RNA from translation because of presence of matching double stranded RNA sequences. RNA interference is a system within the living cells that help to control which genes are active and how active they are. The two small RNA molecules are (a) micro RNA (miRNA) (b) small interfering RNA (siRNA) are central to RNA interference.
RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer , which binds and cleaves double stranded RNA's (ds RNA) to produce ds-fragments of 25 - 25 base pairs with few unpaired overhang bases on each end. It was earlier known that both sense and antisense RNA could silence the gene. As both could cause silencing, the mechanism could not just be pairing of antisense RNA to mRNA and coined the term RNA interference for the unknown mechanism.
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RNA interference is a vital part of the of the immune response to virus and other foreign genetic material, especially plants where it may also prevent self-propagation by transposons. Plants such as Arabidopsis thaliana express multiple dices homologs that are specialized to react differently when the plant is exposed to different types of virus . Even before the RNAi pathway was fully understood it was known that induced gene silencing in plants in a systemic effect and could be transferred from stock top scion plants via grafting.. This feature of plant adaptive immune system allows the entire plant to respond to virus after an initial localized encounter . Many plant viruses evolved with elongated mechanisms to suppress the RNAi response in plant cells .
Generally animals express fewer variants of the dices enzyme than the plants but in some animals RNAi showed antiviral response. In both Juvenile and adult Drosophila, RNA interference is important in antiviral innate immunity and is active against pathogens such as Drosophila X virus . The role of RNA interference in mammalian innate immunity is poorly understood and relatively little data is available , but the existence of virus that encodes genes are capable of suppressing the RNAi response to mammalian cells which favour RNAi - dependent mammalian immune response.
As for the discovery of the RNAi therapy, the following are the following are the consequences that summoned up.
RNAi protects against viral infection 
RNAi secures genome stability by keeping mobile elements silent .
RNAi like mechanisms repress protein synthesis and regulate the development of organisms .
RNAi like mechanisms keep chromatin condensed and suppress transcription.
RNAi offers a new experimental tool to repress genes specifically .
RNAi might be useful in future gene therapy as it is possible to achieve RNAi governed gene regulation in transgenic organisms , that has stimulated many explorations of whether this would be an useful option in medical therapy. Many promising results have been reported in several animal models .
Problems associated with delivery of large molecules:
Virus as vectors:
There is no other organism that has very high very high rate of delivering DNA into our cells other than virus. This feature makes it an advantage to use it for introducing foreign gene into the genome which in turn delivers this gene of interest into human cells at a very high efficient rate.
Many virus have been used such as Adenovirus, Adeno-associated virus and HSV. Many trials are going on to make them safer without any side effects.
Herpes simplex virus:
The most common virus that is known to cause cold sores (HSV). Some of the unique properties of this virus help to study as vector in therapy for central nervous system diseases.. The lifelong infection makes this virus useful as a therapy vector in nervous system with long term gene expression. DNA genome and numerous dispensable genes allows cloning of large transgenes and their regulatory sequences .These vectors can be non replicating or replicating vectors and have minimum amount of virus sequences suitable for delivery up to 150kbp of foreign DNA . The wild type (HSV) and HSV vectors can cause innate and adaptive immune response in host organism .
HSV has relatively large double stranded linear DNA genome within a capsid which is enveloped in a lipid bilayer. This complete particle is called virion . HSV contains atleast 74 genes within their genome .
Figure 1: structure of HSV.
The nucleo capsid is composed of 2 capsomere forms (a) hexons (b) pentons which are connected by heterotrimeric complex called triplex . VP5 is the major capsid protein (149 kDa) which is present both inpentons and hexons. Five copies of which are present in penton (11-12 per capsid) and six copies are present in hexons (150 per capsid). Pentons and hexons are joined 320 triplexes with the help of one copy of VP19c (150 kDa) and two copies of VP23 (34 kDa) .
There are two ways of using HSV as vector:
The process in which the gene of interest is introduced into the plasmid containing HSV origin of replication and packaging signal. This complex is then introduced into cells by transfection and is helped by helper HSV .
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This method id the introduction of gene of interest into a genome. As in the below figure(2) we can see how the gene is cloned into the plasmid vector
Figure2: Introduction of foreign DNA into HSV-1 genome by recombination
When the plasmid is transfected along with HSV-DNA, recombination will take place between viral sequences with plasmid and sequences in the virus genome. This causes introduction of foreign genome into HSV genome .jjjj
The major advantage of HSV is that it infects human neuron cells naturally to produce asymptomatic latent infection unlike other (A-AV, Adenovirus) which infects when injected directly .
Retrovirus have been identified in many agents ranging from eukaryotes to humans. Their replication is identified and characterized by enzyme reverse transcriptase(RT). This enzyme converts the genetic information into DNA intermediate. This virus is of great interest because they are associated important diseases including cancers, AIDS and neurological diseases. Endogenous retrovirus (ERVs) are present in most Eukaryotic organisms and represent retroviral infection into germ cells during evolution . Retrovirus have the advantage of stably integrating their genomes into target cells which made it to develop them as a therapy vector . Retrovirus are classified into Exogenous and Endogenous depending upon their mode of transmission. Exogenous retrovirus infect somatic cells but not germline cells and are transmitted via infecton .
Endogenous retrovirus (ERVs) infect by integrating into germline cells. 10% of mammal genomes are occupied by ERV and are mostly inactivated by deletion and mutation of stop codon.
Figure 3:- structure of retrovirus.
Retroviral vector is made from the proviral form. They have 5' - 3' - LTRs and packaging signal which is required for packaging the RNA genome into retroviral particles . It contains single stranded RNA molecule as the genome. The virions are 80-100nm in diameter containing linear, single stranded, non-segmented DNA of 7-12 Kb in length. Reverse transcription method is involved for the conversion of viral RNA into linear double-stranded DNA and also the integration of this dsDNA into the genome of the host cells . This integrated state is known as pro-virus. This provirus will later undergo transcription and translation in the same manner as cellular genes to produce genomic viral RNA and mRNAs that code for viral proteins. Assembly of new retroviral particles will bud from the host cell to infect other cells .