Gene therapy is an experimental disease which involves fight or prevents disease into human body by express DNA at t site in vivo for long enough and produce therapeutic response which is useful to treat number of disease such as Parkinson's disease, haemophilia, cancer. Gene can be delivered to cell by carrier called 'vectors' such as 'viruses'. Limitations of gene therapy as therapeutics agents, such as poor bioavailability, stability, delivery to the site of action, apply to conventional medicines. 
Basic approaches in gene therapy:
Replacing a mutated gene with healthy gene
Inactivation or knock out of mutated gene
Introducing new gene into body which help fight a disease
There are two types of carrier:
 Viral vectors: Viruses are inserted in as genetic material into the host cell which is part of replication cycle and has instructions to produce more copies of these types of viruses and hijack the body's normal production process. (Shown in figure 1). The host cells will carry out production of the viruses which lead to more cells become infected. Some types of cells insert their genes into the host genomes which incorporates the genes of that virus among the genes of host cell for the life span of that cell. Genes which produce disease in the virus, that is removed and that gege will replace with gene coding. Click to play video in new window
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 Non viral vectors: direct application of the transgene to the target tissue. Non viral therapy has certain advantages over viral methods, with simple large scale production and low host immunogenetically. Most common non viral therapies are- Naked DNA, Oligonucleotides gene therapy.
Clinical problems using gene delivery:
Gene delivery, Targeting, specificity and selectivity
Gene delivery can be achieved by using of vector which may be either viral or non-viral, the ideal viral should be safe and technically easy to produce in large quantities but it must be deliver to specific target. 
Targeting can be achieved either at gene transduction level or gene expression level. Simplest way is injection of material directly into tumour. Sometimes viral vectors and liposomal surfaces can be modifying to bear ligands which bind to specific tissue. 
Antisense Oligonucleotide therapy (ASO):
Antisense therapy involves the administration of synthetic oligonucleotides that are complementary to specific mRNA transcripts. Oligonucleotides must be incorporated in cell and active at nanomolar to micro molar concentrations.
Inside the cell, ASO hybridizes to the specific molecules target mRNA to form DNA or RNA. Ribonuclease H (RNase H) recognises DNA/RNA, cleaves mRNA strand and renders the message non-translatable. Specific mRNA fragments are destroyed by ribonucleases. As a result of this target proteins are diminished.
Oligonucleotides are digested by phophodiaster so they should stabilise against phophodiaster (PD) and most common way is to replace oxygen atoms in phophodiaster with sulphur atom and to form phosphorothioate (PS) from oligonucleotides. [5, 6] PS oligos inhibit gene expression by hybridization arrest which is followed by cleavages of mRNA and which may produce unspecific effects.
Mechanism of action: ASO must reach at cell and remain sufficient concentration in cell for produce biological effects. At concentration below 1µ mol/L, uptake of PS oligonucleotides is predominant receptor like Mechanism and at higher concentration, fluid phase endocytosis mechanism.
Figure.2 Activation of RNase H
Oligonucleotides have been reported to the endocytosed via clathrin-coated. Intracellular oligonucleotides may escape endosome or lysosomes compartment and move into the nucleus. (7-8)
ASO makes pairs with specific sequences of unstructured region of mRNA which is suitable for substrate of RNase H, which cleaves RNA and open way for further degradation (9). In the intracellular target, oligos specifically binds to the sequence of interest and inactivate mRNA target. Inactivation can be due to either sequestration of message or cleavage of mRNA. (10)
Clinical problems of antisense therapy:
ASO have some problems when using in mediated disease, viral disease, cancer, like degradation by nucleolytic enzymes and difficulty to cross cell membrane. To avoid this problem, modification of oligonucleotides has been done. Replacement of one of the non-bridging oxygen atoms at each phosphorus by methyl group has been done but it reduces antisense activity.  Other option is that modification of sulphur atom for oxygen atoms at each phosphorus which generate phosphorothioate (PS ASO). . PS ASO may have non-specific effects either due to nonspecific sequence binding or activation of nonspecific mechanisms. The efficacy of ASO in inhibiting gene expression is depend on ability to penetrate target cells which is depend on temperature, structure and concentration of oligonucleotides.
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RNAi is an innate cellular process that directs the degradation of mRNA homologous to short double stranded RNA (dsRNA).
Small interfering RNA (siRNA) is short double started RNA of 21-23 nucleotides which are able to hybridize with complementary mRNA and induce specific degradation. This mechanism called RNA interference and most efficient siRNA compose 21-nucleotides sense and antisense strand at the 3'ends, both present two nucleotides. 
By Dicer (RNAse III), large size double stranded RNA are intracellulary divide into small parts called siRNA. A protein complex associate with RNA induced silencing complex (RISC) which eliminates one of the two strand siRNA and interact with target RNA through the sequence of complementary which is given by RNA fragment. Argonaute 2 endonuclease cleaves the hybridized mRNA, which is associated with RISC. Degradation of cleaved siRNA will be done by cytoplasmic exonucleases. RISC may be involved in cleaves of mRNA that is a last property which allows gene extinction. 
There are three ways to the introduction of siRNA in cytoplasm through cleavage of long dsRNA by Dicer
Chemically synthesised siRNA into cytoplasm by passing first step (Dicer cleavages)
Use plasmids or viral vectors either two strands or small stem-loop called haiepin RNA
Action of endogenous non coding small RNA[17-18]
Figure 4. Modalities of generation or short RNA
Synthesis siRNAs by Dicer
Long dsRNA are proceed by Dicer into siRNA
Perfect duplex hairpin which need to be cleaved by Dicer
Imperfect duplex hairpin RNAs
Clinical problems in RNAi therapeutics:
RNAi therapeutics has better specifity, potency and versatility but challenges are Delivery of RNAi, safety and efficacy.
RNAi can be either delivered as 'drug' or as DNA encoding hairpin RNA (shRNA). shRNA delivered to cells which allows for intracellular expression of shRNA which proceed into siRNA by host cell.
Safety of any therapeutics aim is to maximize the ratio of desired effects to undesired effects. RNAi has advantage of specificity so it has capacity to provide better gene targeting. Undesired effects can be reducing by controlling off target effects, non specific effects and saturation of RNAi machinery.
Efficacy of RNAi therapeutics can be controlled by avoiding resistance and incomplete inhibiton, prolong duration of action and induce stability of RNAi. 
Ribozymes are RNA molecules that catalyze biochemical reactions. Ras protein plays important roles in signal transduction pathway. Ras transduces the signal from cell surface to the nuclease where genes signal turned on or off through incoming signal. Ribozymes cleave single-stranded regions in RNA through transe-sterification or hydrolysis reactions that result in cleavage of phophodiaster bonds.The hammerhead ribozymes is composed of two regions: a catalytic core effecting cleavage and two flanking sequences confirming Binding and specificity. The hammerhead ribozymes can inhibit target gene expression by several mechanisms. The ribozymes can either be delivered to a cell as RNA construct or as Plasmid which are code for Ribozymes .
Clinical problems in Ribozymes:
In treatment of cancer cells, alteration of signal transduction has been reported. Especially ras gene mutation has been found in 90% of pancreatic adenocarcinomas, so ras genes become targets of gene modulation. Problem of delivery to appropriate targets cells has to be overcome for success of ribozymes mediated gene therapy. 
In HIV, target site, delivery of ribozymes and efficacy are several problems. Efficacy of ribozymes needs to improve by several strategies-
Multi targeted ribozymes were targeted either into shotgun or tandem ribozymes
Combination of anti HIV ribozymes with RNA decoys
Utilisation of anti HIV ribozymes with long antisense RNA