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Gene Therapy:

Lokesh Singhal Department of Pharmaceutical Sciences BBA Central University Gene Therapy 1


Genes Are carried on a chromosome The basic unit of heredity Encode how to make a protein DNA RNA proteins Proteins carry out most of life’s function. When altered causes dysfunction of a protein When there is a mutation in the gene, then it will change the codon, which will change which amino acid is called for which will change the conformation of the protein which will change the function of the protein. Genetic disorders result from mutations in the genome. 2

Picture of a Chromosome:

Picture of a Chromosome 3

What is Gene Therapy:

What is Gene Therapy It is a technique for correcting defective genes that are responsible for disease development There are four approaches: A normal gene inserted to compensate for a nonfunctional gene. An abnormal gene traded for a normal gene An abnormal gene repaired through selective reverse mutation Change the regulation of gene pairs 4

The Beginning…:

The Beginning… In the 1980s, Scientists began to look into gene therapy. They would insert human genes into a bacteria cell. Then the bacteria cell would transcribe and translate the information into a protein Then they would introduce the protein into human cells 5

The First Case:

The First Case The first gene therapy was performed on September 14 th , 1990 Ashanti DeSilva was treated for SCID Sever combined immunodeficiency Doctors removed her white blood cells, inserted the missing gene into the WBC, and then put them back into her blood stream. This strengthened her immune system Only worked for a few months  6

Severe Combined Immunodeficiency Disease (SCID):

Severe Combined Immunodeficiency Disease (SCID) Before GT, patients received a bone marrow transplant received BM from his sister  unfortunately he died from a a form of blood cancer SCID is caused by an Adenosine Deaminase Deficiency (ADA) Gene is located on chromosome #22 Deficiency results in failure to develop functional T and B lymphocytes 7

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ADA is involved in purine degradation Accumulation of nucleotide metabolites = TOXIC to developing T lymphocytes B cells don’t mature because they require T cell help Patients cannot withstand infection  die if untreated 8

Different Delivery Systems are Available:

Different Delivery Systems are Available In vivo versus ex vivo In vivo = delivery of genes takes place in the body Ex vivo = delivery takes place out of the body, and then cells are placed back into the body

In vivo techniques usually utilize viral vectors :

In vivo techniques usually utilize viral vectors Virus = carrier of desired gene Virus is usually “crippled” to disable its ability to cause disease Viral methods have proved to be the most efficient to date Many viral vectors can stable integrate the desired gene into the target cell’s genome

Ex vivo manipulation techniques :

Ex vivo manipulation techniques Electroporation Liposomes Calcium phosphate Gold bullets (fired within helium pressurized gun) Retrotransposons (jumping genes – early days) Human artificial chromosomes

In vivo techniques viral vectors How It Works:

In vivo techniques viral vectors How It Works A vector delivers the therapeutic gene into a patient’s target cell The target cells become infected with the viral vector The vector’s genetic material is inserted into the target cell Functional proteins are created from the therapeutic gene causing the cell to return to a normal state 12

Picture :

Picture  13


Viruses Replicate by inserting their DNA into a host cell Gene therapy can use this to insert genes that encode for a desired protein to create the desired trait Four different types 14

(1) Retroviruses:

(1) Retroviruses Created double stranded DNA copies from RNA genome The retrovirus goes through reverse transcription using reverse transcriptase and RNA the double stranded viral genome integrates into the human genome using integrase integrase inserts the gene anywhere because it has no specific site May cause insertional mutagenesis One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division) 15

(2) Adenoviruses:

(2) Adenoviruses Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans Not replicated though  Has to be reinserted when more cells divide Ex. Common cold 16

Adenovirus cont.:

Adenovirus cont. 17

(3) Adeno-associated Viruses:

(3) Adeno-associated Viruses Adeno-associated Virus- small, single stranded DNA that insert genetic material at a specific point on chromosome 19 From parvovirus family- causes no known disease and doesn't trigger patient immune response. Low information capacity hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding. Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year 18

(4) Herpes Simplex Viruses:

(4) Herpes Simplex Viruses Double stranded DNA viruses that infect neurons Ex. Herpes simplex virus type 1 19

Non-viral Options:

Non-viral Options Direct introduction of therapeutic DNA But only with certain tissue Requires a lot of DNA Creation of artificial lipid sphere with aqueous core, liposome Carries therapeutic DNA through membrane Chemically linking DNA to molecule that will bind to special cell receptors DNA is engulfed by cell membrane Less effective  Trying to introduce a 47th chromosome Exist alongside the 46 others Could carry a lot of information 20

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Genetic Defects that are Candidates for Gene Therapy 21

Current Status:

Current Status FDA hasn’t approved any human gene therapy product for sale Reasons: In 1999, 18-year-old Jesse Gelsinger died from multiple organ failure 4 days after treatment for omithine transcarboxylase deficiency. Death was triggered by severe immune response to adenovirus carrier January 2003, halt to using retrovirus vectors in blood stem cells because children developed leukemia-like condition after successful treatment for X-linked severe combined immunodeficiency disease 22

Problems with Gene Therapy:

Problems with Gene Therapy Short Lived Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time Would have to have multiple rounds of therapy Immune Response new things introduced leads to immune response increased response when a repeat offender enters Viral Vectors patient could have toxic, immune, inflammatory response also may cause disease once inside Multigene Disorders Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because you need to introduce more than one gene May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis 23

Recent Developments:

Recent Developments Genes get into brain using liposomes coated in polymer call polyethylene glycol potential for treating Parkinson’s disease RNA interference or gene silencing to treat Huntington’s siRNAs used to degrade RNA of particular sequence abnormal protein wont be produced Create tiny liposomes that can carry therapeutic DNA through pores of nuclear membrane Sickle cell successfully treated in mice THANKS 24

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