Gene Therapy Dr Hanan Hussien Shehata Nonviral Options

Gene Therapy Dr. Hanan Hussien Shehata

Non-viral Options Alternatives being considered are complexes of DNA with lipids and proteins.

Transfection The term is often used for nonviral methods in eukaryotic cells Its present meaning of a change in cell properties caused by introduction of DNA.

Transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).

Non-viral Options Simplest method of non-viral transfection is direct DNA injection. Clinical trials to inject naked DNA plasmids have been performed successfully. There have been trials with naked PCR products, which have had greater success. Research efforts have yielded several nonviral methods

Non-viral Options (Transformation) 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 47 th chromosome ◦ Exist alongside the 46 others ◦ Could carry a lot of information ◦ But how to get the big molecule through membranes?

Ex vivo manipulation techniques Examples: ◦ ◦ ◦ DEAE-dextran Electroporation Liposomes Calcium phosphate Human artificial chromosomes

DEAE-dextran The positively charged DEAE-dextran molecule interacts with the negatively charged phosphate backbone of the nucleic acid. The DNA–DEAE dextran complexes appear to adsorb onto the cell surface and be taken up by endocytosis. The advantages of this technique are its relative simplicity and reproducibility of results.

Calcium Phosphate The principle involves mixing DNA in a phosphate buffer with calcium chloride. The resulting calcium-phosphate–DNA complexes adhere to the cell membrane and enter the cytoplasm by endocytosis.

Electroporation The use of high-voltage pulses to introduce DNA into cultured cells. Cells in a suitable cuvette are subjected to a short high-voltage pulse that causes the membrane potential of the cells to break down. As a result, pores are formed through which macromolecules such as DNA can enter.

Cationic Liposome The liposomes currently in use typically contain a mixture of cationic and neutral lipids organized into lipid bilayer structures. Transfection-complex formation is based on the interaction of the positively charged liposome with the negatively charged phosphate groups of the nucleic acid. The uptake of the liposome–DNA complexes may be mediated by endocytosis.

Other Methods Sonoporation (ultrasonic frequencies to disrupt cell membrane) Magnetofection (use of magnetic particle Gene guns (shoots DNA coated gold particles into Receptor mediated complexed with DNA) cells by using high pressure) Each method has its own advantages and disadvantages.

47 th artificial chromosome ◦ Researchers are also experimenting with introducing a 47 th artificial chromosome to the body. Could carry a lot of information It would exist autonomously along side of the other 46, not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic information and the body’s immune system would not attack it. However, these processes are still inefficient, are limited to ex-vivo gene transfer

Target tissues for gene therapy Hematopoietic cells derived from bone marrow (BM) may be readily obtained and manipulated ex-vivo in a variety of tissue culture system. Furthermore, therapies based on transfer of genetically modified hematopoietic cells are potentially applicable to a wide range disorders, including the hemoglobinopathies, AIDS and cancer. For these reasons, BM cells are attractive targets in gene therapy research.

Target tissues for gene therapy Gene transfer into human hematopoietic stem cells capable of proliferating in-vivo for long period of time, giving rise to large number of progeny expressing the desired product, remains an elusive goal.

Target tissues for gene therapy The known regenerative properties of the liver make it an attractive gene transfer target. Disorders theoretically amenable to this form of therapy include familial hypercholesterolemia, hemophilia, urea cycle defect, -1 -antitrypsin deficiency and phenylketonuria.

Target tissues for gene therapy Other potential targets for therapy include skeletal muscle cells for treatment of muscular dystrophies, epithelial cells respiratory for the treatment of respiratory insufficiency in cystic fibrosis, and central nervous system tissue for degenerative neurologic disorders.

Limitations, Drawbacks or problems 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 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

Limitations, Drawbacks or problems of Gene Therapy Patient safety Expense ◦ 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 ? ? ? or May induce a tumor if integrated in a tumor suppressor gene because of insertional mutagenesis ◦ 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

The Beginning… More than 5000 patients have been treated in last ~12 years worldwide In the 1980 s, 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

Severe Combine Immunodeficiency ◦ Severe combined immunodeficiency is due to a deficiency of adenosine deaminase, 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

Severe Combine Immunodeficiency Continued…… A previous attempt at gene therapy for immunodeficiency was successful in children with severe combined immunodeficiency. In these patients, peripheral T cells were transduced with a vector bearing the gene for adenosine deaminase. The experiment was extremely labor intensive, because done on mature peripheral -blood T cells, and the procedure therefore had to be repeated many times to achieve success.

Why ADA? 1. The disease is caused by a defect in a single gene. 2. The gene is regulated in a simple, “always on” fashion, unlike many genes whose regulation is complex. 3. The amount of ADA present does not need to be regulated precisely. Even small amounts are beneficial, and large amounts are well tolerated.

Other Examples of Gene Therapy: Single Gene Defects = Most Attractive Candidates Cystic fibrosis AIDS Familial Hypercholesterolemia Lesch-Nyhan Disease Gaucher’s disease

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