PHR 403 Pharmaceutical Biotechnology Gene Therapy Course Instructor Md. Samiul Alam Rajib Senior Lecturer, Department of Pharmacy BRAC University

Overview of the chapter Concept of Gene and genetic disorders Ex vivo versus in vivo gene therapy Potential target diseases for gene therapy Gene transfer methods Viral gene delivery systems Non viral gene delivery systems Anti-sense technology

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.

Genetic disorders Chromosomal disorders Single Gene disorders Gametes have abnormal chromosome numbers and mutations Offspring inherit extra chromosome or are missing a chromosome Caused by problems with meiosis

Genetic disorders Chromosomal disorders: Nondisjunction of chromosomes during meiosis Based on: Brooks/Cole – Thomson Learning

Genetic disorders Chromosomal disorders: Example: Down Syndrome Down Syndrome Characteristics: wide, rounded face enlarged tongue Lower cognitive ability equal length fingers webbed neck Normal female karyotype with 46 chromosomes Down syndrome karyotype with extra chromosome 21

Concept of Inheritance Important points of inheritance: Genes have different forms, called alleles Each trait is controlled by effects of two alleles Some alleles are: Dominant/recessive Other alleles are co-dominant

Concept of Inheritance Case: Hairline trait Allele for Widow’s Peak is dominant Allele for Straight Hairline is recessive A person with WW or Ww will have Widow’s Peak A person with ww will have a Straight Hairline

Concept of Inheritance Genotype Vs Phenotype Genotype refers to the alleles Phenotype refers to the appearance Genotype Phenotype WW person has a widow’s peak Ww ww person has a straight hair line

Genetic disorders Single gene disorders: About 4,000 human diseases are thought to be inherited. Scientists are making good progress figuring out where genes are located on chromosomes. Genetic diseases are caused by Mutations incorrect sequences in the normal form of the gene.

Genetic disorders and human diseases Huntington’s disease: Results in a loss of muscle control and mental function. The symptoms usually do not appear until after 30 years old. 1 in 10,000 people. Caused by a dominant allele

Genetic disorders and human diseases Cystic Fibrosis: Mucus in bronchi is thick, interfering with lung function 1 in 25 are carriers One of the first disorders to be actively studied for gene therapy. Most lethal autosomal recessive disorder in U.S.

Genetic disorders and human diseases

Genetic disorders and human diseases Sickle Cell anemia: Red blood cells are sickle shaped, issues with circulation causing anemia and pain

Genetic disorders and human diseases Sickle cell anemia: Co-dominant disorder found in African Americans. 1 in 400 African Americans Can be fatal. Possible cure: bone-marrow transplants The sickle cell trait can prevent Malaria

Genetic disorders and human diseases Hemophilia: A disorder in which a person’s blood does not clot properly. It is a recessive sex-linked, X-chromosome disorder. 1 in 10,000 males born are afflicted. “Royalty Disease”

Genetic disorders and human diseases Adenoside Deaminase (ADA) Deficiency: ADA-deficient persons are affected by severe immunodeficiency, with recurrent infections that might be life-threatening. First disease approved for gene therapy. Autosomal recessive disorder. The drug exists but is very expensive, needs to be injected in vein for life.

What is gene therapy? Many medical conditions result from flaws, or mutations, in one or more of a person's genes. So, if a flawed gene caused our "broken window," can you "fix" it? What are your options? Imagine that you accidentally broke one of your neighbor's windows. Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed. Repair it with some tape: not the best long-term solution. Put in a new window: not only do you solve the problem, but also you do the honorable thing. Stay silent: ignore the genetic disorder and nothing gets fixed. Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution. Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem!

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 (Gene transplantation). An abnormal gene traded for a normal gene (Gene transplantation) An abnormal gene repaired through selective reverse mutation (Gene correction) Change the regulation of gene pairs (Gene augmentation)

Diseases for applying gene therapy Target Cells Severe Combined Immunodeficiency (SCID) Bone marrow Cells or T-Lymphocytes Hemophilia Liver, Muscle Cystic fibrosis Lung cells Cancer Many cell types Neurological diseases (Parkinson’s, Alzheimer's) Nerve Cells Infectious diseases (AIDS, Hepatitis B) White Blood Cells

in vivo Versus ex vivo Gene Therapy

In vivo Gene Therapy 1. The genetic material is transferred directly into the body of the patient 2. More or less random process; small ability to control; less manipulations 3. Only available option for tissues that can not be grown in vitro; or if grown cells can not be transferred back

ex vivo Gene Therapy The genetic material is first transferred into the cells grown in vitro 2. Controlled process; Genetically altered cells are selected and expanded; more manipulations 3. Cells are then returned back to the patient

Gene delivery systems Non viral gene delivery systems 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 But how to get the big molecule through membranes?

Gene delivery systems Viral gene delivery systems. Retro virus Adenovirus Adeno associated viruses (AAV) Herpes simplex virus (HSV)

Non viral gene delivery systems Direct introduction of therapeutic DNA:

Non viral gene delivery systems Current attempts with naked DNA vaccination in infectious diseases: HIV Hepatitis Influenza Tuberculosis Lyme disease Malaria

Non viral gene delivery systems Direct introduction therapeutic DNA: Ballistic DNA injections: Inventyed for gene transfer in plant cells Fully applicable to eukayotic cells plasmid DNA shown here

Non viral gene delivery systems Gene delivery using Liposomes: Next level idea – why naked DNA? Lets’ wrap it in something safe to increase transfection rate Lipids – are an obvious idea ! Therapeutic DNA

Non viral gene delivery systems Gene delivery using Liposomes: Cheaper than viruses No immune response Especially good for in-lung delivery (cystic fibrosis) 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector

Viral gene delivery systems 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 Retro virus Adenovirus Adeno associated viruses (AAV) Herpes simplex virus (HSV)

Viral gene delivery systems By Retrovirus: 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) vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety

Viral gene delivery systems By Adenovirus: Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans The inserted DNA is not incorporate into genome Not replicated though  Has to be reinserted when more cells divide Ex. Common cold

Viral gene delivery systems (Using Adenovirus)

Viral gene delivery systems By Adeno associated virus: 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 gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. 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

Viral gene delivery systems By Herpes Simplex virus Type 1: Double stranded DNA viruses that infect neurons Ex. Herpes simplex virus type 1

Anti sense therapy Theory: By introducing an antisense gene (or asRNA) into cells, the asRNA would “zip up” the complementary mRNA into a dsRNA that would not be translated The “antisense effect” was highly variable, and in light of the discovery of RNAi, asRNA probably inhibited its target by inducing RNAi rather than inhibiting translation. Used from ~1980 on, to repress specific genes Alternative to gene knock-outs, which were/are very difficult to do in higher plants and animals

Anti sense therapy Mechanism: An antisense oligonucleotide (ASO) is a single-stranded, chemically modified DNA-like molecule that is 17–22 nucleotides in length and designed to be complementary to a selected gene’s mRNA and thereby specifically inhibit expression of that gene Therefore, the level of the target protein is reduced by blocking translation and by altering the subsequent cascades regulating cellular proliferation, differentiation, homeostasis and apoptosis

Anti sense therapy Several mechanisms that explain how ASO inhibit translation have been proposed- 1. The most accepted involves the formation of an mRNA–ASO duplex formed through Watson–Crick binding, leading to RNase-H-mediated cleavage of the target mRNA. 2. Other proposed mechanisms include prevention of mRNA transport, modulation or inhibition of splicing, translational arrest, and formation of a triple helix through ASO binding to duplex genomic DNA resulting in inhibition of transcription. 3. Recent advancements: siRNA

Anti sense therapy

Current scenario 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

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

For the Curious minds!! There is a very recent advancement in Genome editing (in 2014) The technology is called CRISPR/ Cas9 technology A video documentary is uploaded in TSR in case you are interested