GENE THERAPY Medical biotechnology

1. GENE THERAPY Medical biotechnology MaheenAlam 12-1011

2. Objectives • To be able to learn and understand new techniques being used in Gene therapy • To see the basic differences between protien and gene therapy • To be able to learn the ethical issues related to gene therapy • To be able to understand the treatment HIV as an example of gene therapy

3. 1) New approaches to gene therapy • In conventional treatments of gene therapy viral and non-viral vectors are commonly used for the delivery of the gene. • These are used to deliver normal copies of a gene into a cell that tends to contain mutated copies of a gene. • However there are times that when you do add the good copy of the gene it might not work.

4. Dominant negative: • For example there are certain cases when a mutated gene might produce a protein that prevents the normal protein from doing its job and in this case if you simply add the normal gene it won’t help. Mutated genes that work this way are called dominant negative.

6. How do we then deal with a dominant negative? • In this situation one could either repair the product of the mutated gene or they could get rid of it altogether. • Some new methods have been developed by scientists which serve as potential approaches to gene therapy. • Every technique being used for this purpose requires an efficient and specific means of delivering the gene to the target cells. • Some of these are • SMaRT • Triple-helix forming oligonucleotides • Antisense • Ribozymes

7. A technique for repairing mutations: SMaRT: • SMaRT stands for spliceosome-mediated RNA Trans-splicing. • This technique tends to target and repair the messenger RNA transcripts that have been copied from the mutated gene. • Instead of replacing the entire gene this technique tends to repair a particular section of the mRNA that contains the mutation.

8. SMaRT involves three steps • Delivery of a RNA strand that pairs specifically with the intron next to the mutate segment of mRNA. Once bound, this RNA strand prevents spliceosomes from including the mutated segment in the final, spliced RNA product. • Simultaneous delivery of a correct version of the segment to replace the mutated piece in the final mRNA product • Translation of the repaired mRNA to produce the normal, functional protein

9. Techniques to prevent production of a mutated protein: Triple-helix forming oligonucleotides • Triple-helix-forming oligonucleotide gene therapy targets the DNA sequence of a mutated gene to prevent its transcription. • This technique involves the delivery of short, single-stranded pieces of DNA, called oligonucleotides, that bind specifically in the groove between the double strands of the mutated gene's DNA. • Binding produces a triple-helix structure that prevents that segment of DNA from being transcribed into mRNA.

11. Antisense • Antisense gene therapy aims to turn off a mutated gene in a cell by targeting the mRNA transcripts copied from the gene. • Antisense gene therapy involves the following steps: • Delivery of an RNA strand containing the antisense code of a mutated gene • Binding of the antisense RNA strands to the mutated sense mRNA strands, preventing the mRNA from being translated into a mutated protein

13. Ribozymes Like antisense, ribozyme gene therapy aims to turn off a mutated gene in a cell by targeting the mRNA transcripts copied from the gene. This approach prevents the production of the mutated protein. • Ribozyme gene therapy involves the following steps: • Delivery of RNA strands engineered to function as ribozymes. • Specific binding of the ribozyme RNA to mRNA encoded by the mutated gene • Cleavage of the target mRNA, preventing it from being translated into a protein

14. 2) Protein Therapy vs. Gene Therapy

15. There are still serious, unsolved problems related to gene therapy including: 1. Difficulty integrating the therapeutic DNA (gene) into the genome of target cells 2. Risk of an undesired immune response 3 Potential toxicity, immu­nogenicity, inflammatory responses and oncogenesis related to the viral vectors; and 4. The most commonly occurring disorders in humans such as heart disease, high blood pressure, diabetes, Alzheimer’s disease are most likely caused by the combined effects of variations in many genes, and thus injecting a single gene will not be beneficial in these diseases.

16. The benefits of protein therapy include: • Using a human protein with no immuno­genic response • No need for viral vectors • Localized effect at the target tissue, and • Predictability of dose.

17. On the other hand, an obstacle of protein therapy is the mode of delivery: oral, intrave­nous, intra-arterial, or intramuscular routes of the protein’s administration are not always as effective as desired; the therapeutic protein can be metabolized or cleared before it can enter the target tissue. •  It seems that protein therapy will become the treatment modality of choice for many disorders for at least the next 10 years—at least until further research has resolved the hurdles and risks related to gene therapy.

18. 3) Ethical and Social Concerns in Germ-line Gene Therapy • Many unique technical and ethical considerations have been raised by this new form of treatment • Several levels of regulatory committees have been established to review each gene therapy clinical trial prior to its initiation in human subjects. 

19. Ethical considerations include • deciding which cells should be used • how gene therapy can be safely tested and evaluated in humans • what components are necessary for informed consent • and which diseases and/or traits are eligible for gene therapy research.

20. Germ line gene therapy is difficult as stable integration and gene expression requires gene replacement or repair; however currently only gene addition can be done. • Gene addition could result in insertionalmutations and productions of chimeras • Genetic enhancement is another issues which could be misused by totalitarian governments • Also as it tends to be expensive only a certain class can avail the treatment. • The treatment can cause unintended consequences and might affect evolution to a greater degree. • Germ line modifications tend to pose a risk to future generations.

21. 4) Double whammy gene therapy clears HIV from body (Phase I study in 2011) • This study was conducted on 6 patients in California • A person with HIV who didn't take antiretroviral drugs for three months remained free of the virus, thanks to a groundbreaking gene therapy. • The success raises the prospect of keeping HIV in check permanently without antiretrovirals. • The gene therapy works by locking the virus out of the CD4 white blood cells it normally infects. • In this small phase I study they had one virus-free patient and 10-fold reductions in another two.

22. Zinc fingers: • To deliver the treatment, doctors remove blood from the patient and isolate CD4 and other white blood cells. • Specialised molecular "scissors" called zinc finger proteins enter the cells and sabotage a gene called CCR5, which makes a protein that helps HIV to enter cells. • It is unclear what role CCR5plays normally, although researchers know that cells can survive without it – and will remain uninfected by HIV. • These cells are then returned to the patient in the hope that they will multiply and provide a permanent source of cells immune to HIV, potentially locking out HIV completely.

23. Double sabotage • The secret to making the treatment work best, according to research, is therefore to eliminate both genes that make CCR5 in as many cells as possible. If only one is sabotaged, cells can still make enough CCR5 protein to allow the virus to invade. In doubly sabotaged or "bi-allelic" cells, there is no way in.