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Gene therapy

Gene therapy involves using genes as pharmaceutical agents to treat disease. There are four main approaches to gene therapy: inserting a normal gene to compensate for a nonfunctional one, suppressing abnormal gene expression, repairing an abnormal gene through selective reverse mutation, or changing gene regulation. Gene therapy aims to cure the underlying causes of diseases, whereas conventional therapies usually just relieve symptoms. Gene therapy also has the potential for permanent or inheritable effects, unlike most conventional therapies. Common methods for delivering genes include viral vectors like retroviruses, adenoviruses, and AAV, as well as non-viral methods like naked DNA, electroporation, and liposomes.

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Gene Therapy Gene therapy is the use of DNA pharmaceutical agent to treat disease as a
Gene therapy Gene therapy can be broadly defined as the transfer of genetic material into a cell to transiently or permanently alter the cellular phenotype. Molecular surgery
What is Gene Therapy • It is a technique for correcting defective genes that are responsible for disease development • There are four approaches: 1. A normal gene inserted to compensate for a nonfunctional gene. 2. An abnormal gene expression suppressed (antisense Tech) 3. An abnormal gene repaired through selective reverse mutation 4. Change the regulation of gene pairs
Gene Therapy Vs Conventional Therapy Gene Therapy Conventional Therapy Materials DNA, RNA; Cells, Tissues, Or Organs. Small molecules, Peptide, Proteins. Delivery Usually required to be delivered into cells (antisense ODN) or Nucleus (genes). Effect on the cell membrane or diffuse into cells Mechanisms Usually cure the causes of the diseases Usually relieve the symptoms or signs Duration of Effect Can be permanent and also can be passed down to next generation in germline gene therapy. Usually stop the effect once stop taking it. Ethics Major Issues Usually Not
Purpose & approach of gene therapy: • Monogenic gene therapy • Provides genes to encode for the production of a specific protein • Cystic fibrosis, Muscular dystrophy, Sickle cell disease, Haemophilia, SCID • Suicide gene therapy • Provide ‘suicide’ genes to target cancer cells for destruction • Cancer • Antisense gene therapy • Provides a single stranded gene in an’antisense’ (backward) orientation to block the production of harmful proteins • AIDS/HIV
Barriers Of Gene Delivery
Strategies for Transgene Delivery Ex Vivo Cells removed from body Transgene delivered Cells cultured Cells returned to the body In Vivo Transgene delivered directly into host
Gene Therapy Principles AAV Nucleus Adenovirus Therapeutic Protein Retrovirus/Lentivirus Naked DNA Target Cell
Types of vectors for gene delivery • RNA viruses (Retroviruses) 1. Murine leukemia virus (MuLV) 2. Human immunodeficiency viruses (HIV) 3. Human T-cell lymphotropic viruses (HTLV) • DNA viruses 1. Adenoviruses 2. Adeno-associated viruses (AAV) 3. Herpes simplex virus (HSV) 4. Pox viruses 5. Foamy viruses • Non-viral vectors 1. Liposomes 2. Naked DNA 3. Liposome-polycation complexes 4. Peptide delivery systems
Viral Vectors: Gene + Protein Coat • Disabled viral vectors – Genes that cause disease are removed – Gene of interest is inserted • Altered virus should transfer helpful genes to cells but should not multiply or produce disease • Viruses bind to the cell surface receptors of cell membrane and deliver its genetic contents – Do DNA viruses, RNA viruses or both enter the nucleus? • The cell will use the inserted gene to produce a therapeutic protein
Retrovirus for gene delivery
1. Modified Retroviruses (RNA viruses) (1 of 2) Advantages • Good at inserting genes into host chromosome - Used with partial success treating Gaucher’s disease Successfully cured 4 babies of S.C.I.D.S. in early 2000 • Severe Combined Immunodeficiency Syndrome (Bubble Baby)
1. Modified Retroviruses (RNA viruses) (2 of 2) Disadvantages 1. Inserts genes randomly. Possible Consequences? 2. Usually needs an actively dividing host cell • Therefore, not used for Cystic Fibrosis 1. Modified virus may mutate and cause serious disease.
3-D visualization of retrovirus structure.
Life cycle of a retrovirus Gene therapy constructs maintained at this stage.
Retrovirus genome Encapsidation (packaging) 16
Retrovirus genome Encapsidation (packaging) Retrovirus vector construction for gene therapy 5’ LTR Packaging Gene X Neor 3’ LTR 18
Engineering a virus into a viral vector http://www.edu365.com/aulanet/comsoc/Lab_bio/simulacions/GeneTherapy/GeneTherapy.htm Therapeutic Packaging gene Vector DNA wildtype virus Viral vector
Gene transfer Y vector Vector uncoating Episomal vector Target cell Integrated expression cassette Therapeutic mRNA and protein
Adenovirus
Adenovirus particle structure: • Nonenveloped particle • Contains linear double stranded DNA • Does not integrate into the host genome • Replicates as an episomal element in the nucleus
Herpes Simplex Virus Advantages • Large insert size • Could provide long- term CNS gene expression • High titer Disadvantages • System currently under development • Current vectors provide transient expression • Low transduction efficiency
Non-viral vectors 1. Liposomes 2. Naked DNA
Naked DNA • Biolistics now used routinely. DNA coated particles are literally blasted into cells by an explosive discharge. • Electroporation • Pronuclear microinjection 26
‘Particle Gun’ 27
‘Particle Gun’ • DNA coated on pellets is forced down the barrel of a ‘Particle Gun’ by an explosive charge • The particles are forced through the cell wall where the DNA is released Petri Dish with cultures Explosive Charge Projectile DNA coated pellets Barrel Vent Stop plate
Nano particles for gene delivery The electrostatically coated poly(beta-amino ester) nanoparticles can facilitate ligand-mediated gene delivery.
The more promising polymers for gene delivery is degradable poly(beta-amino ester), 1,3-diaminopentaneterminated poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32-117). This polymer functions by binding to and protecting DNA from degradation, enabling efficient cellular uptake, and enabling subsequent intracellular endosomal escape. However, as with many nanoparticle formulations, its systemic use in vivo is limited due to poor biodistribution and lack of tissue-specific targeting
Cationic nanoparticles are formed by first complexing poly (b-amino ester) C32-117 with plasmid DNA at a 30:1 polymer:DNA weight/weight ratio (w/w). These nanoparticles are then coated with poly(glutamic acid)-based peptides (poly-E or poly-E-cat) at 2.5:1–20:1 peptide:DNA w/w. Variation in peptide w/w tunes the biophysical properties of the nanoparticles and subsequent localization of gene delivery by the nanoparticles in vivo.
Pronuclear microinjection of DNA
Electroporation
What is electroporation? • A short controlled pulse of electricity to cell momentarily disrupting lipid bilayer. • Small pores (40-120nm) reseal quickly. Cell wall Nucleu s DNA enters
Electroporation
Electroporation • Use of high-voltage electric shocks to introduce DNA into cells • Cell membranes: electrical capacitors unable to pass current • Voltage results in temporary breakdown and formation of pores Harvest cells and resuspend in electroporation buffer Add DNA to cell suspension…for stable transfection DNA should be linearized, for transient the DNA may be supercoiled electroporat e Selection process for transfectant
This electroporator is for low-current applications such as those using small electrodes
Ex vivo Electroporation
Liposomes
Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material. A net positive charge on this aggregrate has been assumed to increase the effectiveness of transfection through the negatively charged phospholipid bilayer. This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE dextran, calcium phosphate, and electroporation. The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility, and low toxicity.
Lipofection (or liposome transfection)
Example:
Gene therapy for silencing un wanted gene expression Antisense technology
Antisense technology A single-stranded RNA or DNA molecule that is complementary to a target mRNA pairs with the mRNA and prevents translation. This strategy works well in the laboratory on cultured cells and on model organisms. Clinical example: treatment against cancers. The tumor sizes decreased but this was mainly due to the production of interferons in response to high doses of foreign RNA. If the dose was lowered to prevent the interferon response, the clinical benefits largely disappeared as well.
Antisense technology
SiRNA
SiRNA is small interfering RNA. It is also abbreviated as RNAi – RNA intereference. 1.Long double stranded RNA’s will be cleaved by an enzyme called Dicer (endoribonuclease) into short double stranded fragments (-20-25 nucleotides) called siRNA 2. Double stranded siRNA will then be separated into single stranded RNA’s. one strand is called the “guide strand” and the other “Passenger strand”. The guide strand will further bind to RISC (RNA-induced silencing complex) and the passenger strand is degraded. Sometimes, RISC can also be called as RITS (RNA-induced transcriptional silencing). Originally, thought to be an ATP-dependent helicase, which is responsible for unwinding and degradation of the passenger strand, however, later on found to be ATPindependent and the protein components of the RISC does this. 3. Activated RISC complex locates complementary mRNA’s within the cell. 4. Now, this siRNA+RISC complex will go and bind to the complementary bases in the mRNA strand of the targeted gene. “Argonaute”, the catalytic components (protein components) of the RISC will then causes the targeted mRNA strand to cleave, therefore blocking the protein synthesis.
How does siRNA work ?????? (fig.cox.miami.edu/.../gene/how_siRNA_works.htm)
Inherited Disease
Inherited Disease A large number of diseases are known to be inherited from the parents to the offspring. Such diseases are known as Inherited Diseases. A large number of diseases are known to be inherited from the parents to the offspring. Such diseases are known as genetic diseases. Most of these diseases are caused by the expression of recessive genes. The genetic diseases can be broadly classified into two types: •Autosomal disorders •Allosomal disorders Autosomal Disorders: These are metabolic disorders caused by the expression of some genes present on somatic chromosomes. Such disorders express equally in both the sexes. Allosomal Disorders: hese disorders are caused by genes present on the sex chromosomes. The abnormal disorders express more commonly in males than females
Gene therapy for inherited diseases are Severe Combined Immunodeficiency Disease Ornithine transcarbamylase (OTC) deficiency Familial Hypercholesterolemia Cystic Fibrosis Thalassemia Lesch-Nyhan syndrome Hunter’s syndrome Sickle cell trait and Sickle cell anemia
Severe Combined Immunodeficiency Disease (SCID) • SCID is caused by an Adenosine Deaminase Deficiency (ADA) – Gene is located on chromosome #22 (32 Kbp, 12 exons) – Deficiency results in failure to develop functional T and B lymphocytes – ADA is involved in Adenine degradation – Lack of ADA leads to a 100-fold increase in the cellular concentration of dATP, a strong inhibitor of ribonucleotide reductase . – High levelsof dATP produce a general deficiency of other dNTPs in T lymphocytes. – 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
The first case for gene therapy in the world is SCID AMP IMP ADA gene mutation adenosine ADA inosin e adenosine dAMP (adenosine deaminase ) ADA dADP dATPInhibit nucleotide reductase T, B cell proliferation (-) Severe Combined ImmunoDeficiency
Severe Combined Immunodeficiency Disease (SCID) • September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial – Ashanti (4 year old girl) • Her lymphocytes were gene-altered (~109) ex vivo  used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene (billions of retroviruses used) – Cynthia (9 year old girl) treated in same year • Problem: WBC are short-lived, therefore treatment must be repeated regularly
Retrovirus used to deliver gene for Adenosine deaminase Gene therapy constructs maintained at this stage.
Ornithine transcarbamylase (OTC) deficiency – Ornithine transcarbamylase (OTC) deficiency • Urea cycle disorder (1/10,000 births) • Encoded on X chromosome – Females usually carriers, sons have disease – Urea cycle = series of 5 liver enzymes that rid the body of ammonia (toxic breakdown product of protein) • If enzymes are missing or deficient, ammonia accumulates in the blood and travels to the brain (coma, brain damage or death)
Ornithine transcarbamylase (OTC) deficiency • Severe OTC deficiency – Newborns  coma within 72 hours • Most suffer severe brain damage • ½ die in first month • ½ of survivors die by age 5 – Early treatment • Low-protein formula called “keto-acid” – Modern day treatment • Sodium benzoate and another sodium derivative • Bind ammonia  helps eliminate it from the body
Disorders Associated with Defects in Receptor Proteins Familial Hypercholesterolemia • This commonly results from an autosomal dominant defect in a gene for the LDL receptor or receptor function. • At least 900 mutations have been identified affecting different aspects of LDL uptake, metabolism and regulation. • De-novo cholesterol synthesis is normally suppressed by exogenous cholesterol intake; with receptor processing defects this function is lost and markedly elevated cholesterol levels result. • Cholesterol levels are elevated to such an extent that atherosclerotic disease resulting in fatal cardiovascular events beginning in the second & third decades .
There are five major classes of FH due to LDLR mutations: – Class I: LDL receptor (LDL-R) is not synthesized at all – Class II: LDL-R is not properly transported from the endoplasmic reticulum to the Golgi apparatus for expression on the cell surface – Class III: LDL-R does not properly bind LDL on the cell surface ( this may be caused by a defect in either Apolipoprotein B100 or a defect in LDL-R – Class IV: LDL-R bound to LDL does not properly cluster in clathrincoated pits for receptor-mediated endocytosis – Class V: the LDL-R is not recycled back to the cell surface
• Major issue is LDL receptor mutation • This data base shows all the different mutations • For Familial hypercholesterolemia there are 806 mutations • 457 mutations are missense and nonsense
Substitution mutations • • • • GGG-AGG Gly-Arg Hypercholesterolaemia GCG-GAG Ala-Glu Hypercholesterolaemia CTC-CCC Leu-Pro Hypercholesterolaemia cGAG-TAG Glu-Term Hypercholesterolaemia
– Gene Therapy for Familial Hypercholesterolemia – 1993  First attempt • Retroviral vector used to infect 3.2 x 109 liver cells (~15% of patients liver) ex vivo – Infused back into patient – Improvement seen – Has been used in many trials since then
Cystic Fibrosis
Gene therapy for Cystic Fibrosis • Cystic fibrosis (CF) is inherited as an autosomal recessive disease • CF affects the epithelial cells lining air passages to the lungs • CF causes a buildup of mucus in the airways
Clinical Features • Classic cystic fibrosis is characterized by chronic bacterial infection of the airways and sinuses, fat maldigestion due to pancreatic exocrine insufficiency, infertility in males due to obstructive azoospermia, and elevated concentrations of chloride in sweat. • Patients with nonclassic cystic fibrosis have at least one copy of a mutant gene that confers partial function of the CFTR protein, and such patients usually have no overt signs of maldigestion because some pancreatic exocrine function is preserved.
Gene therapy for Cystic Fibrosis • In CF, there is a defective ion channel protein = cystic fibrosis transmembrane conductance regulator (CFTR) • CFTR regulates the balance of Chloride ions in epithelial cell membranes • Patients with Cystic Fibrosis make an altered version of this protein – Protein is misfolded – What types of proteins are involved in helping other proteins fold properly?
Gene therapy for Cystic Fibrosis • Adenovirus vector was used to deliver a normal ion channel protein to airway cells in a patient’s nose or lungs • What is special about adenovirus?
Thalassemia
Gene therapy for thalassemia Thalassemia (also spelled thalassaemia) is an inherited autosomal recessive blood disease. In thalassemia, the genetic defect which could be either mutations or deletion results in reduced rate of synthesis or no synthesis of one of the globin α or βchains that make up hemoglobin. Reduced synthesis or no synthesis of one of the globin chains can cause the formation of abnormal hemoglobin molecules, thus causing anemia, the characteristic presenting symptom of the thalassemias.
The thalassemias are classified according to which chain of the hemoglobin molecule is affected. In α thalassemias, production of the α globin chain is affected, while in β thalassemia production of the β globin chain is affected. β globin chains are encoded by a single gene on chromosome 11; α globin chains are encoded by two closely linked genes on chromosome 16. Thus in a normal person with two copies of each chromosome, there are two loci encoding the β chain, and four loci encoding the α chain. Deletion of one of the α loci has a high prevalence in people of African or Asian descent, making them more likely to develop α thalassemias. β thalassemias are common in Africans, but also in Greeks and Italians. Beta-thalassemia (β-thalassemia) is a form of thalassemia due to mutations in the HBB gene on chromosome 11, inherited in an autosomal recessive fashion. The severity of the disease depends on the nature of the mutation. •Mutations are characterized as (βo) if they prevent any formation of β chains. •Mutations are characterized as (β+) if they allow some β chain formation to occur. Diagnosis: Screening, Pre-natal diagnostics, check for microcytosis (mean cell haemoglobin < 27 pg or mean red cell volume < 80 fl).
Diagnosis of β-thalassemia Deletion by Southern Blotting • Autosomal recessive, decreased or absent β-globin protein. • Mutant alleles have large deletions or point mutations. Restriction Enzyme Cut Sites
Gene therapy for Betathalassemia  Gene transfer of a regulated β-globin gene in HSCs would reduce the imbalance between aand β-globin chains in erythroid cells  Transplantation of autologous, genetically corrected HSCs would represent an alternative therapy for thalassemic patients lacking a suitable bone marrow donor
TERAPIA GENICAfor β-thalassemia Gene therapy DELLA ß-TALASSEMIA 21_11.jpg β-globin vector Purification of CD34+ cells Patient Transduction Infusion of genetically-corrected cells
Lesch-Nyhan syndrome: X-Linked Recessive Disorders (HGRPT deficiency)
 Lesch-Nyhan syndrome condition is inherited in an Xlinked recessive pattern. It mostly affects male, that they have only one X chromosome, thus one altered copy of the gene is sufficient to cause the condition. In females, who have two X chromosomes, a mutation must usually be present in both copies of the gene to cause the disorder.  Lesch-Nyhan syndrome (LNS), also known as Nyhan’s syndrome, is a rare, inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or Kelley-Seegmiller Syndrome that affects the level of uric acid in the body. This disease often affects males. Males with this syndrome develop physical handicaps, mental retardation, and kidney problems. The symptoms of LNS usually appear between the ages of 3 and 6 months.
 The 3 main features of the disease are: Excessive production of uric acid Neurological problems, especially mental retardation and spastic cerebral palsy Behavioral disorders- confusion, anxiety, fear, and obsession
Diagnosis  The diagnosis of Lesch-Nyhan syndrome is based initially on the distinctive pattern of the child's symptoms, most commonly involuntary muscle movements or failure to crawl and walk at the usual ages.  In some cases the first symptom is related to overproduction of uric acid; the parents notice "orange sand" in the child's diapers. The "sand" is actually crystals of uric acid tinged with blood. Measuring the amount of uric acid in a person's blood or urine can not definitively diagnose Lesch-Nyhan syndrome. It is diagnosed by measuring the activity of the HPRT enzyme through a blood test. When the activity of the enzyme is very low it is diagnostic of Lesch-Nyhan syndrome.
Hunter’s syndrome: Xlinked recessive disorder
Hunter’s syndrome, an X-linked recessive disorder. Hunter syndrome, or mucopolysaccharidosis Type II, is a lysosomal storage disease caused by a deficient (or absent) enzyme, iduronate-2-sulfatase. The syndrome is named after physician Charles A. Hunter (1873–1955), who first described it in 1917
Hunter syndrome, or mucopolysaccharidosis II (MPS II), is a serious genetic disorder that primarily affects males (X-linked recessive). It interferes with the body's ability to break down and recycle specific mucopolysaccharides, also known as glycosaminoglycans or GAG. Hunter syndrome is one of several related lysosomal storage diseases. In Hunter syndrome, GAG builds up in cells throughout the body due to a deficiency or absence of the enzyme iduronate-2-sulfatase (I2S). This buildup interferes with the way certain cells and organs in the body function and leads to a number of serious symptoms. As the buildup of GAG continues throughout the cells of the body, signs of Hunter syndrome become more visible.
X-linked recessive Hunter’s syndrome
sickle cell anaemia
The genetics of sickle cell anaemia The shape of the haemoglobin molecule is controlled by two alleles • Normal Haemoglobin allele • Sickle Cell Haemoglobin allele There are three phenotypes Normal Normal individuals have two normal haemoglobin alleles Sickle cell anaemia, a severe form where all the red blood cells are affected. Sickle cell anaemia patients have two sickle cell alleles in their genotypehomozygous Sickle cell trait, a mild condition where 50% of the red blood cells are affected. Sickle cell trait individuals are heterozygotes, having one of each allele
Codominant genotypes Genotypes HbNHbN HbNHbS HbSHbS Phenotypes Normal haemoglobin Sickle cell trait Sickle cell anaemia
The success of gene therapy is based on:  efficient gene transfer into target cells  adequate level of transgene expression  persistence of gene expression  regulation of gene expression  tolerance to transgene product  safety
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
Problems Doing Gene therapy (1 of 2) Inefficient gene delivery—not suitable for all genetic diseases 1. Most effective if Stem cells are involved • • Only to correct a few cells with the gene E.g. Blood stem cells: SCIDS and Gaucher Disease 1. Less effective or Ineffective if many cells must be corrected • • Brain cells (Tay-Sacs disease, Huntington’s disease) Cystic Fibrosis
Problems Doing Gene therapy (2 of 2) 4. Insertion of Gene isn’t always permanent • e.g. Gaucher Disease: temporary cure until GCase gene “popped” out of chromosome 4. Insertion of gene into genome could disrupt other genes. • Possible consequences? 4. Some viruses elicit immune response or may cause disease • E.g. Jesse Gelsinger died in 1999
Gene therapy

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In this document
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Introduction to gene therapy as a DNA-based treatment that alters cell phenotypes. It differs from conventional therapy by targeting disease causes rather than symptoms. Outlines four approaches to gene therapy: gene insertion, suppression, repair, and regulation. Discusses applications in various genetic diseases.

Explains barriers and strategies for gene delivery, focusing on ex vivo and in vivo methods and the use of viral and non-viral vectors.

Details on modified retroviruses, their advantages and disadvantages, and their lifecycle in gene therapy processes.

Describes processes in gene transfer, use of adenoviruses, and non-viral methods like lipofection and biolistics.

Focuses on nanoparticles for gene delivery and their effectiveness in protecting DNA during cellular uptake.

Discusses electroporation for introducing DNA into cells via electrical pulses, detailing the procedure and variations.

Introduces antisense technology and siRNA mechanisms for silencing gene expression and their clinical implications.

Presents inherited diseases and cites specific disorders treatable by gene therapy, including SCID and OTC deficiency.

Discusses gene therapy approaches for genetic diseases like SCID, OTC deficiency, and familial hypercholesterolemia.

Describes cystic fibrosis, its genetic basis, and gene therapy strategies aimed at delivering a normal CFTR gene.

Explains thalassemia's genetic defects impacting hemoglobin synthesis and outlines potential gene therapy approaches.Details on Lesch-Nyhan syndrome and Hunter's syndrome, their genetic causes, symptoms, and implications for gene therapy.

Discusses the genetic underpinnings of sickle cell anemia and reviews challenges and inefficiencies in gene therapy.

Gene therapy

  • 1.
    Gene Therapy Gene therapyis the use of DNA pharmaceutical agent to treat disease as a
  • 2.
    Gene therapy Gene therapycan be broadly defined as the transfer of genetic material into a cell to transiently or permanently alter the cellular phenotype. Molecular surgery
  • 3.
    What is GeneTherapy • It is a technique for correcting defective genes that are responsible for disease development • There are four approaches: 1. A normal gene inserted to compensate for a nonfunctional gene. 2. An abnormal gene expression suppressed (antisense Tech) 3. An abnormal gene repaired through selective reverse mutation 4. Change the regulation of gene pairs
  • 4.
    Gene Therapy VsConventional Therapy Gene Therapy Conventional Therapy Materials DNA, RNA; Cells, Tissues, Or Organs. Small molecules, Peptide, Proteins. Delivery Usually required to be delivered into cells (antisense ODN) or Nucleus (genes). Effect on the cell membrane or diffuse into cells Mechanisms Usually cure the causes of the diseases Usually relieve the symptoms or signs Duration of Effect Can be permanent and also can be passed down to next generation in germline gene therapy. Usually stop the effect once stop taking it. Ethics Major Issues Usually Not
  • 5.
    Purpose & approachof gene therapy: • Monogenic gene therapy • Provides genes to encode for the production of a specific protein • Cystic fibrosis, Muscular dystrophy, Sickle cell disease, Haemophilia, SCID • Suicide gene therapy • Provide ‘suicide’ genes to target cancer cells for destruction • Cancer • Antisense gene therapy • Provides a single stranded gene in an’antisense’ (backward) orientation to block the production of harmful proteins • AIDS/HIV
  • 6.
  • 7.
    Strategies for TransgeneDelivery Ex Vivo Cells removed from body Transgene delivered Cells cultured Cells returned to the body In Vivo Transgene delivered directly into host
  • 8.
  • 9.
    Types of vectorsfor gene delivery • RNA viruses (Retroviruses) 1. Murine leukemia virus (MuLV) 2. Human immunodeficiency viruses (HIV) 3. Human T-cell lymphotropic viruses (HTLV) • DNA viruses 1. Adenoviruses 2. Adeno-associated viruses (AAV) 3. Herpes simplex virus (HSV) 4. Pox viruses 5. Foamy viruses • Non-viral vectors 1. Liposomes 2. Naked DNA 3. Liposome-polycation complexes 4. Peptide delivery systems
  • 10.
    Viral Vectors: Gene+ Protein Coat • Disabled viral vectors – Genes that cause disease are removed – Gene of interest is inserted • Altered virus should transfer helpful genes to cells but should not multiply or produce disease • Viruses bind to the cell surface receptors of cell membrane and deliver its genetic contents – Do DNA viruses, RNA viruses or both enter the nucleus? • The cell will use the inserted gene to produce a therapeutic protein
  • 11.
  • 12.
    1. Modified Retroviruses(RNA viruses) (1 of 2) Advantages • Good at inserting genes into host chromosome - Used with partial success treating Gaucher’s disease Successfully cured 4 babies of S.C.I.D.S. in early 2000 • Severe Combined Immunodeficiency Syndrome (Bubble Baby)
  • 13.
    1. Modified Retroviruses(RNA viruses) (2 of 2) Disadvantages 1. Inserts genes randomly. Possible Consequences? 2. Usually needs an actively dividing host cell • Therefore, not used for Cystic Fibrosis 1. Modified virus may mutate and cause serious disease.
  • 14.
    3-D visualization ofretrovirus structure.
  • 15.
    Life cycle ofa retrovirus Gene therapy constructs maintained at this stage.
  • 16.
  • 18.
    Retrovirus genome Encapsidation (packaging) Retrovirus vectorconstruction for gene therapy 5’ LTR Packaging Gene X Neor 3’ LTR 18
  • 19.
    Engineering a virusinto a viral vector http://www.edu365.com/aulanet/comsoc/Lab_bio/simulacions/GeneTherapy/GeneTherapy.htm Therapeutic Packaging gene Vector DNA wildtype virus Viral vector
  • 20.
    Gene transfer Y vector Vector uncoating Episomalvector Target cell Integrated expression cassette Therapeutic mRNA and protein
  • 21.
  • 22.
    Adenovirus particle structure: •Nonenveloped particle • Contains linear double stranded DNA • Does not integrate into the host genome • Replicates as an episomal element in the nucleus
  • 24.
    Herpes Simplex Virus Advantages •Large insert size • Could provide long- term CNS gene expression • High titer Disadvantages • System currently under development • Current vectors provide transient expression • Low transduction efficiency
  • 25.
  • 26.
    Naked DNA • Biolisticsnow used routinely. DNA coated particles are literally blasted into cells by an explosive discharge. • Electroporation • Pronuclear microinjection 26
  • 27.
  • 28.
    ‘Particle Gun’ • DNAcoated on pellets is forced down the barrel of a ‘Particle Gun’ by an explosive charge • The particles are forced through the cell wall where the DNA is released Petri Dish with cultures Explosive Charge Projectile DNA coated pellets Barrel Vent Stop plate
  • 29.
    Nano particles forgene delivery The electrostatically coated poly(beta-amino ester) nanoparticles can facilitate ligand-mediated gene delivery.
  • 30.
    The more promisingpolymers for gene delivery is degradable poly(beta-amino ester), 1,3-diaminopentaneterminated poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32-117). This polymer functions by binding to and protecting DNA from degradation, enabling efficient cellular uptake, and enabling subsequent intracellular endosomal escape. However, as with many nanoparticle formulations, its systemic use in vivo is limited due to poor biodistribution and lack of tissue-specific targeting
  • 31.
    Cationic nanoparticles areformed by first complexing poly (b-amino ester) C32-117 with plasmid DNA at a 30:1 polymer:DNA weight/weight ratio (w/w). These nanoparticles are then coated with poly(glutamic acid)-based peptides (poly-E or poly-E-cat) at 2.5:1–20:1 peptide:DNA w/w. Variation in peptide w/w tunes the biophysical properties of the nanoparticles and subsequent localization of gene delivery by the nanoparticles in vivo.
  • 32.
  • 33.
  • 34.
    What is electroporation? •A short controlled pulse of electricity to cell momentarily disrupting lipid bilayer. • Small pores (40-120nm) reseal quickly. Cell wall Nucleu s DNA enters
  • 35.
  • 36.
    Electroporation • Use ofhigh-voltage electric shocks to introduce DNA into cells • Cell membranes: electrical capacitors unable to pass current • Voltage results in temporary breakdown and formation of pores Harvest cells and resuspend in electroporation buffer Add DNA to cell suspension…for stable transfection DNA should be linearized, for transient the DNA may be supercoiled electroporat e Selection process for transfectant
  • 37.
    This electroporator isfor low-current applications such as those using small electrodes
  • 38.
  • 39.
  • 40.
    Lipofection (or liposometransfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material. A net positive charge on this aggregrate has been assumed to increase the effectiveness of transfection through the negatively charged phospholipid bilayer. This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE dextran, calcium phosphate, and electroporation. The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility, and low toxicity.
  • 41.
  • 46.
  • 47.
    Gene therapy forsilencing un wanted gene expression Antisense technology
  • 48.
    Antisense technology A single-strandedRNA or DNA molecule that is complementary to a target mRNA pairs with the mRNA and prevents translation. This strategy works well in the laboratory on cultured cells and on model organisms. Clinical example: treatment against cancers. The tumor sizes decreased but this was mainly due to the production of interferons in response to high doses of foreign RNA. If the dose was lowered to prevent the interferon response, the clinical benefits largely disappeared as well.
  • 49.
  • 50.
  • 51.
    SiRNA is smallinterfering RNA. It is also abbreviated as RNAi – RNA intereference. 1.Long double stranded RNA’s will be cleaved by an enzyme called Dicer (endoribonuclease) into short double stranded fragments (-20-25 nucleotides) called siRNA 2. Double stranded siRNA will then be separated into single stranded RNA’s. one strand is called the “guide strand” and the other “Passenger strand”. The guide strand will further bind to RISC (RNA-induced silencing complex) and the passenger strand is degraded. Sometimes, RISC can also be called as RITS (RNA-induced transcriptional silencing). Originally, thought to be an ATP-dependent helicase, which is responsible for unwinding and degradation of the passenger strand, however, later on found to be ATPindependent and the protein components of the RISC does this. 3. Activated RISC complex locates complementary mRNA’s within the cell. 4. Now, this siRNA+RISC complex will go and bind to the complementary bases in the mRNA strand of the targeted gene. “Argonaute”, the catalytic components (protein components) of the RISC will then causes the targeted mRNA strand to cleave, therefore blocking the protein synthesis.
  • 52.
    How does siRNA work?????? (fig.cox.miami.edu/.../gene/how_siRNA_works.htm)
  • 54.
  • 55.
    Inherited Disease A largenumber of diseases are known to be inherited from the parents to the offspring. Such diseases are known as Inherited Diseases. A large number of diseases are known to be inherited from the parents to the offspring. Such diseases are known as genetic diseases. Most of these diseases are caused by the expression of recessive genes. The genetic diseases can be broadly classified into two types: •Autosomal disorders •Allosomal disorders Autosomal Disorders: These are metabolic disorders caused by the expression of some genes present on somatic chromosomes. Such disorders express equally in both the sexes. Allosomal Disorders: hese disorders are caused by genes present on the sex chromosomes. The abnormal disorders express more commonly in males than females
  • 56.
    Gene therapy forinherited diseases are Severe Combined Immunodeficiency Disease Ornithine transcarbamylase (OTC) deficiency Familial Hypercholesterolemia Cystic Fibrosis Thalassemia Lesch-Nyhan syndrome Hunter’s syndrome Sickle cell trait and Sickle cell anemia
  • 57.
    Severe Combined ImmunodeficiencyDisease (SCID) • SCID is caused by an Adenosine Deaminase Deficiency (ADA) – Gene is located on chromosome #22 (32 Kbp, 12 exons) – Deficiency results in failure to develop functional T and B lymphocytes – ADA is involved in Adenine degradation – Lack of ADA leads to a 100-fold increase in the cellular concentration of dATP, a strong inhibitor of ribonucleotide reductase . – High levelsof dATP produce a general deficiency of other dNTPs in T lymphocytes. – 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
  • 58.
    The first casefor gene therapy in the world is SCID AMP IMP ADA gene mutation adenosine ADA inosin e adenosine dAMP (adenosine deaminase ) ADA dADP dATPInhibit nucleotide reductase T, B cell proliferation (-) Severe Combined ImmunoDeficiency
  • 59.
    Severe Combined Immunodeficiency Disease(SCID) • September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial – Ashanti (4 year old girl) • Her lymphocytes were gene-altered (~109) ex vivo  used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene (billions of retroviruses used) – Cynthia (9 year old girl) treated in same year • Problem: WBC are short-lived, therefore treatment must be repeated regularly
  • 60.
    Retrovirus used todeliver gene for Adenosine deaminase Gene therapy constructs maintained at this stage.
  • 61.
    Ornithine transcarbamylase (OTC) deficiency –Ornithine transcarbamylase (OTC) deficiency • Urea cycle disorder (1/10,000 births) • Encoded on X chromosome – Females usually carriers, sons have disease – Urea cycle = series of 5 liver enzymes that rid the body of ammonia (toxic breakdown product of protein) • If enzymes are missing or deficient, ammonia accumulates in the blood and travels to the brain (coma, brain damage or death)
  • 63.
    Ornithine transcarbamylase (OTC) deficiency •Severe OTC deficiency – Newborns  coma within 72 hours • Most suffer severe brain damage • ½ die in first month • ½ of survivors die by age 5 – Early treatment • Low-protein formula called “keto-acid” – Modern day treatment • Sodium benzoate and another sodium derivative • Bind ammonia  helps eliminate it from the body
  • 64.
    Disorders Associated withDefects in Receptor Proteins Familial Hypercholesterolemia • This commonly results from an autosomal dominant defect in a gene for the LDL receptor or receptor function. • At least 900 mutations have been identified affecting different aspects of LDL uptake, metabolism and regulation. • De-novo cholesterol synthesis is normally suppressed by exogenous cholesterol intake; with receptor processing defects this function is lost and markedly elevated cholesterol levels result. • Cholesterol levels are elevated to such an extent that atherosclerotic disease resulting in fatal cardiovascular events beginning in the second & third decades .
  • 65.
    There are fivemajor classes of FH due to LDLR mutations: – Class I: LDL receptor (LDL-R) is not synthesized at all – Class II: LDL-R is not properly transported from the endoplasmic reticulum to the Golgi apparatus for expression on the cell surface – Class III: LDL-R does not properly bind LDL on the cell surface ( this may be caused by a defect in either Apolipoprotein B100 or a defect in LDL-R – Class IV: LDL-R bound to LDL does not properly cluster in clathrincoated pits for receptor-mediated endocytosis – Class V: the LDL-R is not recycled back to the cell surface
  • 68.
    • Major issueis LDL receptor mutation • This data base shows all the different mutations • For Familial hypercholesterolemia there are 806 mutations • 457 mutations are missense and nonsense
  • 69.
    Substitution mutations • • • • GGG-AGG Gly-ArgHypercholesterolaemia GCG-GAG Ala-Glu Hypercholesterolaemia CTC-CCC Leu-Pro Hypercholesterolaemia cGAG-TAG Glu-Term Hypercholesterolaemia
  • 70.
    – Gene Therapyfor Familial Hypercholesterolemia – 1993  First attempt • Retroviral vector used to infect 3.2 x 109 liver cells (~15% of patients liver) ex vivo – Infused back into patient – Improvement seen – Has been used in many trials since then
  • 71.
  • 72.
    Gene therapy forCystic Fibrosis • Cystic fibrosis (CF) is inherited as an autosomal recessive disease • CF affects the epithelial cells lining air passages to the lungs • CF causes a buildup of mucus in the airways
  • 73.
    Clinical Features • Classiccystic fibrosis is characterized by chronic bacterial infection of the airways and sinuses, fat maldigestion due to pancreatic exocrine insufficiency, infertility in males due to obstructive azoospermia, and elevated concentrations of chloride in sweat. • Patients with nonclassic cystic fibrosis have at least one copy of a mutant gene that confers partial function of the CFTR protein, and such patients usually have no overt signs of maldigestion because some pancreatic exocrine function is preserved.
  • 74.
    Gene therapy forCystic Fibrosis • In CF, there is a defective ion channel protein = cystic fibrosis transmembrane conductance regulator (CFTR) • CFTR regulates the balance of Chloride ions in epithelial cell membranes • Patients with Cystic Fibrosis make an altered version of this protein – Protein is misfolded – What types of proteins are involved in helping other proteins fold properly?
  • 75.
    Gene therapy forCystic Fibrosis • Adenovirus vector was used to deliver a normal ion channel protein to airway cells in a patient’s nose or lungs • What is special about adenovirus?
  • 76.
  • 77.
    Gene therapy for thalassemia Thalassemia(also spelled thalassaemia) is an inherited autosomal recessive blood disease. In thalassemia, the genetic defect which could be either mutations or deletion results in reduced rate of synthesis or no synthesis of one of the globin α or βchains that make up hemoglobin. Reduced synthesis or no synthesis of one of the globin chains can cause the formation of abnormal hemoglobin molecules, thus causing anemia, the characteristic presenting symptom of the thalassemias.
  • 78.
    The thalassemias areclassified according to which chain of the hemoglobin molecule is affected. In α thalassemias, production of the α globin chain is affected, while in β thalassemia production of the β globin chain is affected. β globin chains are encoded by a single gene on chromosome 11; α globin chains are encoded by two closely linked genes on chromosome 16. Thus in a normal person with two copies of each chromosome, there are two loci encoding the β chain, and four loci encoding the α chain. Deletion of one of the α loci has a high prevalence in people of African or Asian descent, making them more likely to develop α thalassemias. β thalassemias are common in Africans, but also in Greeks and Italians. Beta-thalassemia (β-thalassemia) is a form of thalassemia due to mutations in the HBB gene on chromosome 11, inherited in an autosomal recessive fashion. The severity of the disease depends on the nature of the mutation. •Mutations are characterized as (βo) if they prevent any formation of β chains. •Mutations are characterized as (β+) if they allow some β chain formation to occur. Diagnosis: Screening, Pre-natal diagnostics, check for microcytosis (mean cell haemoglobin < 27 pg or mean red cell volume < 80 fl).
  • 79.
    Diagnosis of β-thalassemiaDeletion by Southern Blotting • Autosomal recessive, decreased or absent β-globin protein. • Mutant alleles have large deletions or point mutations. Restriction Enzyme Cut Sites
  • 80.
    Gene therapy forBetathalassemia  Gene transfer of a regulated β-globin gene in HSCs would reduce the imbalance between aand β-globin chains in erythroid cells  Transplantation of autologous, genetically corrected HSCs would represent an alternative therapy for thalassemic patients lacking a suitable bone marrow donor
  • 81.
    TERAPIA GENICAfor β-thalassemia Genetherapy DELLA ß-TALASSEMIA 21_11.jpg β-globin vector Purification of CD34+ cells Patient Transduction Infusion of genetically-corrected cells
  • 82.
    Lesch-Nyhan syndrome: X-Linked RecessiveDisorders (HGRPT deficiency)
  • 83.
     Lesch-Nyhan syndromecondition is inherited in an Xlinked recessive pattern. It mostly affects male, that they have only one X chromosome, thus one altered copy of the gene is sufficient to cause the condition. In females, who have two X chromosomes, a mutation must usually be present in both copies of the gene to cause the disorder.  Lesch-Nyhan syndrome (LNS), also known as Nyhan’s syndrome, is a rare, inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or Kelley-Seegmiller Syndrome that affects the level of uric acid in the body. This disease often affects males. Males with this syndrome develop physical handicaps, mental retardation, and kidney problems. The symptoms of LNS usually appear between the ages of 3 and 6 months.
  • 84.
     The 3main features of the disease are: Excessive production of uric acid Neurological problems, especially mental retardation and spastic cerebral palsy Behavioral disorders- confusion, anxiety, fear, and obsession
  • 85.
    Diagnosis  The diagnosisof Lesch-Nyhan syndrome is based initially on the distinctive pattern of the child's symptoms, most commonly involuntary muscle movements or failure to crawl and walk at the usual ages.  In some cases the first symptom is related to overproduction of uric acid; the parents notice "orange sand" in the child's diapers. The "sand" is actually crystals of uric acid tinged with blood. Measuring the amount of uric acid in a person's blood or urine can not definitively diagnose Lesch-Nyhan syndrome. It is diagnosed by measuring the activity of the HPRT enzyme through a blood test. When the activity of the enzyme is very low it is diagnostic of Lesch-Nyhan syndrome.
  • 86.
    Hunter’s syndrome: Xlinkedrecessive disorder
  • 87.
    Hunter’s syndrome, anX-linked recessive disorder. Hunter syndrome, or mucopolysaccharidosis Type II, is a lysosomal storage disease caused by a deficient (or absent) enzyme, iduronate-2-sulfatase. The syndrome is named after physician Charles A. Hunter (1873–1955), who first described it in 1917
  • 88.
    Hunter syndrome, ormucopolysaccharidosis II (MPS II), is a serious genetic disorder that primarily affects males (X-linked recessive). It interferes with the body's ability to break down and recycle specific mucopolysaccharides, also known as glycosaminoglycans or GAG. Hunter syndrome is one of several related lysosomal storage diseases. In Hunter syndrome, GAG builds up in cells throughout the body due to a deficiency or absence of the enzyme iduronate-2-sulfatase (I2S). This buildup interferes with the way certain cells and organs in the body function and leads to a number of serious symptoms. As the buildup of GAG continues throughout the cells of the body, signs of Hunter syndrome become more visible.
  • 89.
  • 90.
  • 91.
    The genetics ofsickle cell anaemia The shape of the haemoglobin molecule is controlled by two alleles • Normal Haemoglobin allele • Sickle Cell Haemoglobin allele There are three phenotypes Normal Normal individuals have two normal haemoglobin alleles Sickle cell anaemia, a severe form where all the red blood cells are affected. Sickle cell anaemia patients have two sickle cell alleles in their genotypehomozygous Sickle cell trait, a mild condition where 50% of the red blood cells are affected. Sickle cell trait individuals are heterozygotes, having one of each allele
  • 92.
  • 93.
    The success ofgene therapy is based on:  efficient gene transfer into target cells  adequate level of transgene expression  persistence of gene expression  regulation of gene expression  tolerance to transgene product  safety
  • 94.
    Problems with GeneTherapy • 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
  • 95.
    Problems Doing Genetherapy (1 of 2) Inefficient gene delivery—not suitable for all genetic diseases 1. Most effective if Stem cells are involved • • Only to correct a few cells with the gene E.g. Blood stem cells: SCIDS and Gaucher Disease 1. Less effective or Ineffective if many cells must be corrected • • Brain cells (Tay-Sacs disease, Huntington’s disease) Cystic Fibrosis
  • 96.
    Problems Doing Genetherapy (2 of 2) 4. Insertion of Gene isn’t always permanent • e.g. Gaucher Disease: temporary cure until GCase gene “popped” out of chromosome 4. Insertion of gene into genome could disrupt other genes. • Possible consequences? 4. Some viruses elicit immune response or may cause disease • E.g. Jesse Gelsinger died in 1999

Editor's Notes

  • #4 Is the most common approach The abnormal gene would be swapped by homologous recombination Would cause a return to normal function Control expression of genes. Similar to epistasis, when one gene affects the expression of another gene.
  • #37 First used by wong and neumann in fibroblasts, then generalized to many other cell types Resistance of buffers: low resistance is high salt
  • #82 21_11.jpg