Introduction to Gene Therapy

Maulik P. Suthar

2009, Maulik P. Suthar

What is gene therapy? Why is it used?

Gene therapy = Introduction of normal genes into cells that contain defective genes to reconstitute a missing protein product GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized to improve a genetic disorder Gene therapy is a technique for correcting defective genes responsible for disease development.

2009, Maulik P. Suthar

How is Gene Therapy Carried Out?

Modification of somatic cells by transferring desired gene sequences into the genome. Somatic cells necessary to ensure that inserted genes are not carried over to the next generation.

2009, Maulik P. Suthar

Approaches for correcting a genetic defect

A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common. An abnormal gene could be swapped for a normal gene through homologous recombination. The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

2009, Maulik P. Suthar

How does the normal gene replace the abnormal gene?

A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. The most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Target cells such as the patient's liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell.

2009, Maulik P. Suthar

Different Delivery Systems

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

2009, Maulik P. Suthar

Strategies for Transgene Delivery

Ex Vivo
Cells removed from body Transgene delivered Cells cultured

In Vivo
Transgene delivered directly into host

Cells returned to the body

2009, Maulik P. Suthar

Vectors
The way you insert the normal gene in the patients cell is by vectors. The most common vectors that are used in gene therapy are virus vectors

2009, Maulik P. Suthar

Why Viruses?
Viruses through the time of evolution have evolved to infect the cells with great specificity Viruses tend to be very efficient at transfecting their own DNA into the host cell genome. This allows them to produce new viral particles at the period of synthesis of the cell

2009, Maulik P. Suthar

Gene Therapy Principles

AAV Nucleus

Adenovirus
Therapeutic Protein Target Cell
2009, Maulik P. Suthar

Retrovirus/Lentivirus Naked DNA

 2009, Maulik P. Suthar

In vivo techniques
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 cells genome

2009, Maulik P. Suthar

Types of Viral Vectors

Retroviruses - A class of viruses that can create doublestranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus. Adenoviruses - A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus. Adeno-associated viruses - A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19. Herpes simplex viruses - A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.
2009, Maulik P. Suthar

Adenovirus
36 kb Double Stranded DNA Genome Entry through CAR receptor and integrin co-receptor P. Suthar 2009, Maulik

E1A E1B

L1

L2

L3

L4

E3

L5

ITR

Stuffer DNA

Therapeutic Transgene
E2A

Stuffer DNA

ITR
E4

E2B

Latest Generation Adenoviral Vector

Gutless; Helper-dependent; Minimal Ad
2009, Maulik P. Suthar

Which Virus to Use?

Depends
how well they transfer the genes to cells which cells they can recognize and infect and whether they alter the cells DNA permanently or temporarily

2009, Maulik P. Suthar

Non viral methods

Direct introduction of therapeutic DNA into target cells (microinjection). This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA. Artificial liposomes which carry the therapeutic DNA are capable of passing the DNA through the target cell's membrane. Can be non-specific to cell type. Chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options. A 47th artificial human chromosome. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of DNA, and scientists anticipate that, because of its construction and autonomy, the body's immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

2009, Maulik P. Suthar

 Problem: Replication defective viruses adversely affect the virus normal ability to spread genes in the body
Reliant on diffusion and spread Hampered by small intercellular spaces for transport Restricted by viral-binding ligands on cell surface therefore cannot advance far.

2009, Maulik P. Suthar

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

2009, Maulik P. Suthar

Limitations of Gene Therapy

Gene delivery Limited tropism of viral vectors Dependence on cell cycle by some viral vectors (i.e. mitosis required) Duration of gene activity Non-integrating delivery will be transient (transient expression) Integrated delivery will be stable

2009, Maulik P. Suthar

Patient safety
Patient safety Immune hyperresponsiveness (hypersensitivity reactions directed against viral vector components or against transgenes expressed in treated cells) Integration is not controlled oncogenes may be involved at insertion point cancer? More than 5000 patients have been treated in last ~12 years worldwide

2009, Maulik P. Suthar

 Gene control/regulation Most viral vectors are unable to accommodate full length human genes containing all of their original regulatory sequences Human cDNA often used much regulatory information is lost (e.g. enhancers inside introns)

2009, Maulik P. Suthar

 Often promoters are substituted therefore gene expression pattern may be very different Random integration can adversely affect expression (insertion near highly methylated heterogeneous DNA may silence gene expression)

2009, Maulik P. Suthar

 Expense Costly because of cell culturing needs involved in ex vivo techniques Virus cultures for in vivo delivery Usually the number of patients enrolled in any given trial is <20

2009, Maulik P. Suthar

Example: Severe Combined Immunodeficiency Disease (SCID)

Before GT, patients received a bone marrow transplant David, the Boy in the Bubble, 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 (32 Kbp, 12 exons) Deficiency results in failure to develop functional T and B lymphocytes

2009, Maulik P. Suthar

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

2009, Maulik P. Suthar

 Severe OTC deficiency Newborns coma within 72 hours Most suffer severe brain damage die in first month 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
2009, Maulik P. Suthar

Other Examples of Gene Therapy: Single Gene Defects = Most Attractive Candidates Cystic fibrosis
Crippled adenovirus selected (non-integrating, replication defective, respiratory virus) Gene therapy trials 3 Research teams, 10 patients/team
2 teams administered virus via aerosol delivery into nasal passages ad lungs 1 team administered virus via nasal passages only Only transient expression observed because adenovirus does not integrate into genome like retroviruses

2009, Maulik P. Suthar

 AIDS HIV patients T lymphocytes treated ex vivo with rev and env defective mutant strains of HIV Large numbers of cells obtained Injected back into patient Stimulated good CD8+ cytotoxic T cell responses (Tcyt)

2009, Maulik P. Suthar

 Familial Hypercholesterolemia Defective cholesterol receptors on liver cells Fail to filter cholesterol from blood properly Cholesterol levels are elevated, increasing risk of heart attacks and strokes 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

2009, Maulik P. Suthar

 Lesch-Nyhan Disease Candidate Early days confined to animal models and in vitro tests Defect in producing HGPRT enzyme (hypoxanthineguanine phosphoribosyl transferase) Defective metabolism of hypoxanthine and guanine Uric acid accumulates Gout, Kidney disease, cerebral palsy, mental retardation, head banging, profanity, spitting, mutilation of fingers

2009, Maulik P. Suthar

 Gauchers disease Glucocerebrosidase gene defect RAC approved clinical tests 1993 Affects CNS, enlarged spleen and liver, long bone erosion and discoloration of skin

2009, Maulik P. Suthar

Gene Therapy of Cancer

Cancer results from multiple mutations Limited success with p53--growth arrest versus apoptosis Target the Rb pathway INK/ARF locus--two potential targets inhibit cyclin PTEN expression alters metastatic potential and reduces vascularization New tumor suppressors such as mda-7 (melanoma) and OPCML(ovarian cancer)

2009, Maulik P. Suthar

Methods for gene therapy of cancer

Viruses Naked DNA (vector-free) Liposomes Protein-DNA complexes Gene gun Calcium phosphate precipitation Electroporation Intracellular microinjection

2009, Maulik P. Suthar

Reasons for lack of clinical success

Low transduction frequency Insufficient expression in vivo

2009, Maulik P. Suthar

Strategies
#1: Strengthening of the immune response against a tumor B7 expression on tumors may provide necessary second signal (costimulation) required for Tcyt cell activation Improve antigen presenting properties of tumor cells Antibodies target tumors for destruction by immune system Monoclonal antibody binding to tumor antigens can stimulate: NK cell killing via antibody-dependent cell-mediated cytotoxicity Phagocytosis via FcR-binding on macrophages Complement activation opsonization

2009, Maulik P. Suthar

 Attract cells of the immune system to the tumor

Example: Transfect tumor cells with cytokine genes GM-CSF Recruits dendritic cells to site of tumor Dendritic cells pick up tumor antigen, process it, and travel to draining lymph nodes to present antigen to T cells

Load professional Antigen Presenting Cells of patient with tumor antigen in vitro and then administer them back to patient
T cells of patient can now recognize tumor cells and will destroy them

2009, Maulik P. Suthar

 #2: Repair of cell cycle defects caused by loss of tumor suppressor gene
e.g. p53 = DNA repair enzyme Guardian of the genome
Faulty p53 allows cells carrying damaged DNA to survive when they would normally die
Mutations passed to progeny Can accumulate additional mutations lethal tumor

In most human cancers, the p53 gene appears defective

2009, Maulik P. Suthar

 #3: Repair of cell cycle defects caused by inappropriate activation of oncogenes

Oncogenes = mutant versions of normal genes (proto-oncogenes) that drive cell growth

2009, Maulik P. Suthar

 Example: ras gene (20-30% of all human cancers have an abnormal ras gene)
Normally = relay switch within the signal pathway that tells the cell to divide In absence of external stimulation ras is off Mutant ras = switch stuck in the on position misinforming cells mitosis

2009, Maulik P. Suthar

Problems to overcome
Short-lived nature of gene therapy Immune response Problems with viral vectors Multigene disorders

2009, Maulik P. Suthar

Current Gene Therapy progress

Into the brain using liposomes coated in a polymer call polyethylene glycol (PEG). RNA interference or gene silencing may be a new way to treat Huntington's. New gene therapy approach repairs errors in messenger RNA derived from defective genes. Technique has potential to treat the blood disorder thalassaemia, cystic fibrosis, and some cancers. Gene therapy for treating children with X-SCID (sever combined immunodeficiency) or the "bubble boy" disease is stopped in France when the treatment causes leukemia in one of the patients. Sickle cell is successfully treated in mice.

2009, Maulik P. Suthar