1. Gene therapy 2 TREATMENT OF GENETIC DISEASE Many genetic disorders are characterized by progressive disability or chronic ill-health for which there.

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Gene therapy

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TREATMENT OF GENETIC DISEASE

• Many genetic disorders are characterized by progressive disability or chronic ill-health for which there is, at present, no effective treatment.

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CONVENTIONAL APPROACHES TO TREATMENT OF GENETIC DISEASE

• Most genetic disorders cannot be cured or even ameliorated using conventional methods of treatment.

• Sometimes this is because the underlying gene and gene product have not been identified so that there is little, if any, understanding of the basic metabolic or molecular defect.

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Treatment of Genetic Disease with non genetically methods

• Substrate limitation (PKU) or hormone replacement, as in congenital adrenal hyperplasia

• Replacement of a deficient product (Thyroid Hormone)

• In a few disorders, such as homocystinuria and some of the organic acidurias, supplementation with a vitamin or coenzyme can increase the activity of the defective enzyme with beneficial effect

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Gene therapy

• Gene therapy involves the direct genetic modification of cells of the patient in order to achieve a therapeutic goal. This modification usually occurs by introducing exogenous DNA using viral vectors or other means

• Gene therapy can be defined as the replacement of a deficient gene product or correction of an abnormal gene.

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The history of gene therapy

• On September 14th, 1990 researchers at the U.S. National Institutes of Health performed the first (approved) gene therapy procedure on four-years old Ashanti DeSilva. Born with a rare genetic disease called severe combined immune deficiency (SCID), she lacked a healthy immune system, and was vulnerable to every passing germ.

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Candidate Diseases for Gene Therapy

• Gene therapy is likely to have the greatest success with diseases that are cause by single gene defects. By the end of 1993, gene therapy had been approved for use on such diseases as severe combined immune deficiency, familial hypercholesterolemia, cystic fibrosis, and Gaucher's disease. Most protocols to date are aimed toward the treatment of cancer; a few are also targeted toward AIDS. Numerous disorders are discussed as candidates for gene therapy: Parkinson's and Alzheimer's diseases, arthritis, and heart diseases

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Target cells for gene therapy

• Germ-line gene therapy: produces a permanent transmissible modification (modification of a gamete, zygote or an early embryo). This method has ethical problems.

• Somatic cell gene therapy: All current gene therapy trials and protocols are for somatic cell therapy.

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Ways of genetic modification of somatic cell :

• Gene supplementation (augmentation); To supply a functioning copy of a defective gene. (C F, Tumor suppressor gene and cytokine)

• Gene replacement (gene targeting); To replace a mutant gene by a correctly functioning copy, or to correct a mutation in situ.

• Targeted inhibition of gene expression; in infectious diseases, cancers, and autoimmune diseases for silencing pathogen gene, oncogenes and Ab production.

• Targeted killing of specific cells; for cancer treatment (TK and nervous system tumors).

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Methods of gene therapy

• Ex vivo gene therapy; Hematopoietic Stem Cell

• In vivo gene therapy; Intravenous injection or injection in solid tissue such as liver or brain.

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Gene therapy methods

Viral vectors

• Gene targeting

• Gene silencing

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Viral vector advantages:

• Viruses are the most commonly used vectors for gene therapy

• Viral vectors are highly efficient vehicles for gene delivery to cells.

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Viral Vectors

• Adenovirus • Aden Associated virus• Retrovirus and Lentivirus• Herpes virus • Vaccinia virus

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• These vectors have high capacity for foreign DNA, wide host range including non-dividing cells (high tropism for epithelial cells), high stability and titer (1011 pfu/ml)

• Gutless adenoviral vectors ( Lack of E1/E4) have low immunological response and large capacity (37kb).treatment of CF and ADA.

Transient expression in dividing cell, Immunologic response in next application , One case of death following an extreme inflammatory response to adenoviral gene therapy.

Adenovirus viral vector

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Adeno-associated viral vectors

• Safe in handling, integrate in genome (chromosome 19), stable transformation, wide host range including non-dividing cells.

In new version rep is deleted and integration is randomly, Low capacity (about 5 kb),

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Herpesvirus vectors

• HSV vectors are particularly suitable for gene therapy in the nervous system , because the virus is remarkably neurotropic.

• Large capacity, Transient(do not integrate)

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Retrovirus vectors• Highly efficient for transduction (about 100%),stable

transformation, third generation of retroviral vectors have broad host range because they have VSV G glycoprotein in their envelops (pseudo type vector), moderate capacity (7-8 kb)

Random integration, Retroviral vector can transduce only dividing cells but lentiviral vectors are able to transduce dividing and non dividing cells

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Gene Therapy with Viral Vectors

Advantages and disadvantage highly efficient for gene delivery Random Integration Oncogene Activation Tumor suppressor gene inactivation Low Titers, instability Transcription silencing Immunogenicity

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Viral vector production kit elements

• A Recombinant plasmid which has expression cassette of interest gene and positive selection marker and sequences for packaging

• Packaging cell line which has coding gene of viral capsid proteins and some essential enzyme

• Final product is a replicating defective virus

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Biological activity assaysBiosafety considerations

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Drug

• An obviously successful example of this is the use of factor VIII concentrate in the treatment of hemophilia A

• injection of the enzyme or protein may not be successful if the metabolic processes involved are carried out within cells and the protein or enzyme is not normally transported into the cell.

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Drug delivery by liposome

• Artificial delivery systems, such as liposomes, allow proteins to cross the cell membrane. Liposomes are artificially prepared cell-like structures in which one or more bimolecular layers of phospholipid enclose one or more aqueous compartments, which can include specific proteins.

• For example, modifications in β-glucosidase as used in the treatment of Gaucher's disease enable it to enter the lysosomes, resulting in an effective form of treatment .

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Tissue Transplantation

• Renal transplantation in adult polycystic kidney disease or lung transplantation in CF

• BMT in hematologic cancer or hemoglobinopathies• Islet cells are prepared from donated pancreases

(usually two per patient) and injected into the liver of the recipient. The 'Edmonton' protocol has proved very successful: 3 years post transplant over 80% of patients are still producing their own insulin

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Drug treatment

• familial hypercholesterolemia (Statins)• avoidance of certain drugs or foods can prevent the

manifestation of the disorder, e.g. sulphonamides and fava beans in glucose-6-phosphate dehydrogenase (G6PD) deficiency

• A recent example is a trial where gentamicin was administered via nasal drops to patients with cystic fibrosis. Aminoglycoside antibiotics such as gentamicin cause readthrough of premature stop codons in vitro and only patients with nonsense mutations showed evidence of expression of full-length CFTR protein in the nasal epithelium.

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Stem-cell transplantation

Bone-marrow transplantation can be an effective treatment for patients with a number of genetic disorders, including ADA deficiency, severe-combined immunodefficiency (SCID), lysosomal storage diseases and Fanconi anemia.

The main limitation is the lack of a suitable bone-marrow donor, but it is hoped that the use of stem cells derived from cord blood may overcome this problem in the future.

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In utero stem cell transplantation

• Trials of in utero stem cell transplantation are under way for a number of disorders that include severe combined immunodeficiency (SCID), α- and β-thalassemia and sickle-cell disease.

• Embryonic stem cells For example, it is possible to culture mouse embryonic stem cells

to generate dopamine-producing neurons. When these neural cells were transplanted into a mouse model for Parkinson disease, the dopamine-producing neurons showed long-term survival and ultimately corrected the phenotype.

• Therapeutic cloning

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THERAPEUTIC APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY

• Biosynthesis of gene products• Insulin from pig• We should clone only amplified cDNA in

prokayotic vector because prokayotes do not have splicing system.

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• biosynthetically derived products have the dual advantages of providing a pure product that is unlikely to induce a sensitivity reaction and one that is free of the risk of chemical or biological contamination.

• In the past, the use of growth hormone from human cadaver pituitaries has been associated with the transmission of Creutzfeldt-Jakob disease, and HIV has been a contaminant in cryoprecipitate containing factor VIII used in the treatment of hemophilia A

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NIH and GTAC

• In the United States the Human Gene Therapy Subcommittee of the National Institutes of Health has produced guidelines for protocols of trials of gene therapy that must be submitted for approval to both the Food and Drug Administration and the Recombinant DNA Advisory Committee, along with their institutional review boards (IRBs).

• In the UK the Gene Therapy Advisory Committee (GTAC) advises on the ethical acceptibility of proposals for gene therapy research in humans, taking account of the scientific merits, and the potential benefits and risks.

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TECHNICAL ASPECTS• Gene characterization by sequencing and

biological activity test• Target cells/tissue/organ: for example

preparation of hematopoietic stem cells from bone marrow by mini MACS device (CD34+ cells)

• Vector system: A safe and potent vector with appropriate regulatory, promoter and enhancer sequences.

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Safety

• it is essential to demonstrate that introduction of the foreign gene or DNA sequence has no deleterious effects through mistakes arising as a result of the insertion of the gene or DNA sequence into the host DNA, or what is known as insertional mutagenesis.

• In both patients who developed leukemia after gene therapy for XL-SCID, the retrovirus used to deliver the γ-c (IL2RG) gene had inserted into the LMO-2 oncogene on chromosome 11, which plays a role in some forms of childhood leukemia.

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METHODS OF GENE THERAPY

• viral methods• non-viral methods.Naked DNAChemical reagent, liposome Electroporation,ultrasound

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Non-viral methods

• These have the theoretical advantage of not eliciting an immune response and being safer and simpler to use, as well as allowing large-scale production, but differ in their efficacy.

• Naked DNA:Direct injection of DNA into cells has been used in gene therapy, e.g. the minidystrophin gene into myoblasts in the mouse model for Duchenne muscular dystrophy

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Liposome-mediated DNA transfer

• An advantage of liposome-mediated gene transfer is that a much larger DNA sequence can be introduced into the target cells or tissues than with viral vector systems.

• This can be as large as an to a specific structural gene like human artificial chromosome

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Receptor-mediated endocytosis

• DNA complexed to a glycoprotein containing galactose will be recognized by receptors on the surface of liver cells that are specific to glycoproteins with a terminal galactose.

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Repair of mutations in situ through the cellular DNA repair machinery

• A promising new approach is the use of chimeric double-stranded DNA/RNA oligonucleotides to repair genes in situ through the cellular DNA repair machinery.

• Proof of principle has been demonstrated in an animal model of Pompe disease, where the point mutation was shown to be repaired at the DNA level and normal enzyme activity restored.

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Antisense-induced exon splicing

• If an exon-splicing enhancer (ESE) is mutated, the exon is more likely to be spliced out. Some proteins with in-frame whole exon deletions retain some residual activity, e.g. dystrophin mutations in Becker muscular dystrophy.

• In an in vitro experiment using muscle cells from two patients with Duchenne muscular dystrophy, it was possible to block an ESE with an antisense oligonucleotide (a short synthetic DNA fragment complementary in sequence to the mRNA).

• The reading frame was restored and the detection of significant levels of dystrophin protein in the muscle cells confirmed the therapeutic potential of this approach.

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In utero fetal gene therapy

• The report of successful adenovirus vector-mediated in utero gene therapy in a cystic fibrosis mouse model in 1997 means that fetal gene therapy in utero may be possible in humans.

• At present it is considered unacceptable because of the possibility of inadvertent germ cell modification.

• The use of stem cells genetically modified ex vivo should reduce this risk.

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Animal models

• One of the basic prerequisites for assessing the suitability of gene therapy trials in humans is the existence of an animal model.

• Animal models for cystic fibrosis, Duchenne muscular dystrophy, Huntington disease, and Friedreich ataxia have been generated and provide just a few examples that may be used to evaluate gene therapy before trials in humans

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There are 3 major classes of nucleic acids-based gene silencing molecules:

• Antisense oligodeoxyribonucleotides• Ribozymes• RNAi (siRNAs and miRNAs)Intrabody

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Antisense technology

• Short (15-mer) oligonucluotides complementary to the target mRNA.• Modified DNA backbone for better uptake, hybridization, stability, etc.• Does not depend on special cellular machinery, and thus works in every

cell type or tissue, in vitro as well as in vivo.• Not complete (“knock-down”).

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Antisense RNA

• RNase H recognizes a 7-base-pair heteroduplex, so the region of homology between the antisense DNA and target mRNA need not be very long.

• Antisense RNA forms double-stranded regions that block either protein translation or splicing of introns.

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Antisense RNA

• Chemically synthesized antisense oligonucleotides are traditionally made of DNA rather than RNA for two reasons. DNA is more stable in the laboratory, and DNA synthesis is an established and automated procedure.

• one compound has already been approved for treatment of cytomegalovirus-induced retinitis

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Ribozyme technology

•A specifically-designed ribozyme cleaves a specific pathogenic RNA molecule to make it inactive. For example, the viral RNA causing hepatitis C.

•Very promising results using cell cultures.

•The ribozyme is synthesized in vitro and administrated to the patient.

Problems: half-life too short and low potency once in the body.

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RNA interference

• In contrast to antisense oligonucleotide therapy, as a result of RNA interference the target mRNA is cleaved rather than just bound, and it is estimated to be up to 1000-fold more active.

• In vitro, siRNAs have been shown to reduce in vitro expression of Bcr-Abl and Bcl2 targets in cancer, viral infections including HIV, and polyglutamine repeat sequences in neurodegenerative disorders.

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RNAi technology

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Intrabodies

• Intracellular antibodies, or intrabodies, are designed to mimic antibody-antigen binding, but within the cell.

• Intrabodies are derived from the V (variable) regions of the heavy and light chains in order to harness the affinity of the parent antibody.

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Two conceptual distinctions are central to an understanding of

the ethical issues of gene therapy • Therapy vs. enhancement ( Therapy not enhancement) There is a

consensus that gene therapy should be therapy, i.e. the correction of bona fide disease conditions, rather than enhancement, which would mean "improving the human species" (whatever that means...) and therefore would entail the introduction in human subjects of novel characteristics going beyond the usual, medical, understanding of health (i.e. health as absence of serious disease).

• Somatic vs. germ line gene therapy All current research on humans deals with somatic gene therapy. In these projects somatic cells such as bone-marrow, liver, lung or vascular epithelium etc. are genetically modified. Since the germ line is not affected, all effects of therapy end with the life of the patient, at the very latest. In fact, most somatic therapies will probably require repeated applications, much like ordinary pharmacological treatments

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Gene therapy projects have followed a manic depressive course over the past 15 years as cycles of optimism are followed by bouts of excessive pessimism. (successful treatment of ADA deficiency, death of Jesse Gelsinger, successful treatment of two SCID children, then they developed leukemia due to gene therapy.

Over 800 trial protocols related to gene therapy have been approved

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Target organs

• In many instances gene therapy will need to be and should be directed or limited to a particular organ, tissue or body system.

• Liver• Viral vectors for gene therapy of inherited hepatic

disorders have been of limited use due to the lack of vectors that specifically target hepatocytes. Although liver cells are refractory to retroviruses in vivo, they are, somewhat surprisingly, susceptible to transfection by retroviruses in vitro.

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Liver

• Cells removed from the liver by partial hepatectomy can be treated in vitro and then reinjected via the portal venous system, from which they seed in the liver.

• The effectiveness of this approach has been demonstrated by the lowering of cholesterol levels in a rabbit animal model with a defect in the low-density lipoprotein (LDL) receptor.

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Liver

• However, associated with a significant risk of thrombosis of the portal venous system that can lead to the complication of portal hypertension.

• Nevertheless, because of the serious outlook for homozygotes with mutations in the LDL receptor , gene therapy by this means has been attempted in a woman homozygous for an LDL receptor defect.

• This has led, in the short term, to a reduction of LDL levels, although the long-term benefit is, as yet, undetermined

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CNS

• Adenoviral and lentiviral vectors• lentiviruses could be used for the treatment of

central nervous system disorders, such as Parkinson and Alzheimer diseases, because they integrate into the host genome of non-dividing cells and could, therefore, act as a delivery system for stable expression.

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CNS

• Another approach which has been suggested in genetic disorders affecting the CNS is to transplant cells that have been genetically modified in vitro into specific regions of the brain, such as the caudate nucleus in persons with or at risk of Huntington disease

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Muscle

• Unlike other tissues, direct injection of foreign DNA into muscle has met with some success in terms of retention and expression of the foreign gene in the treated muscle

• Injection of genetically corrected myoblasts into muscle• Direct DNA injection has, however, been used to

express the protein products of genes, transferred in vitro into myoblasts, which are unrelated to muscle function, such as human growth hormone and factor VIII.

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Bone marrow

• Stem cells often constitute less than 1% of the total cells present in BM aspiration

• Pretreatment of the bone marrow to expand the number of stem cells has been tried for certain inherited immunological disorders by the use of growth factors such as the granulocyte colony-stimulating factor (G-CSF) and the cytotoxic agent 5-fluorouracil.

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Adenosine deaminase deficiency

• The most successful conventional treatment for ADA deficiency is bone-marrow transplantation, but in the absence of a compatible donor, patients may be treated with PWG-conjugated ADA.

• In 1990 the first gene therapy trial enrolled 10 patients with ADA-SCID. Although no adverse events were reported, none of the patients were 'cured', probably due to the low efficiency of gene transfer from the retroviral vector.

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ADA

• A trial is now under way where patients are not be treated with PEG-ADA, but receive mild chemotherapy prior to ex vivo treatment of their bone marrow cells with a novel retroviral vector.

• The chemotherapy aims to kill off diseased cells and enhance the selective growth advantage of the modified cells. Two patients have been successfully treated using this approach

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Hemoglobinopathies

• Attempts at treating β-thalassemia and sickle-cell disease by gene therapy have not been effective as yet, primarily because the numbers of α- and β-globin chains must be equal

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Cystic fibrosis

• Double-blind trials are being carried out in the UK and the USA treating cystic fibrosis patients using either a liposome-gene complex or an adeno-associated virus vector sprayed into the nasal passages. Studies of their efficacy and safety involve looking for the presence of the introduced CFTR gene in biopsies of the nasal epithelium and measurement of ion transport in nasal epithelial cells.

• Reports to date of trials of gene therapy in cystic fibrosis have shown that it is not obviously harmful, although there is very limited evidence of efficacy.

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Hemophilia A and B

• Hemophilia A and B are excellent candidates for gene therapy as a modest increase in the level of factor VIII or IX, respectively, will be of major clinical benefit.

• Recent trials have used direct intramuscular injection of adeno-associated virus expressing factor VIII or ex vivo treatment of fibroblasts with plasmid-borne factor IX followed by injection into the stomach cavity.

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Duchenne muscular dystrophy

• The main difficulty with gene therapy for Duchenne muscular dystrophy is the sheer size of the dystrophin gene (the cDNA is 14kb).

• An alternative strategy is to use antisense oligonucleotides to force exon skipping and convert out-of-frame deletions that cause DMD to in-frame deletions usually associated with the milder Becker muscular dystrophy phenotype.

• There is also the possibility of upregulating a dystrophin homolog, utrophin(drug finding and design for upregulating the expression of utrophin)

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Cancer

• In contrast to the limited number of gene therapy trials for single-gene disorders, numerous cancer-gene therapy trials have been initiated.

• Gene therapy for cancer aims to selectively kill cancer cells either directly through the use of toxins targeted at cancer cells, or by enhancing the body's immune response.

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Supply tumor suppressor genes

• It has been proposed that the targeted introduction of recognized tumor suppressor genes, such as Tp53, to cancer cells could result in control of their growth.

• Inhibit oncogenic proteins • Imatinib (also known as STI-571 or Glivec) is a

protein tyrosine kinase inhibitor used to treat chronic myeloid leukemia. It is a very effective treatment that works by binding the Bcr-Abl fusion protein resulting from the t(9;22) translocation.

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Stimulate natural killing of tumor cells

• Mitogens, such as interleukin-2, introduced in vitro into melanosomes, which have been removed from patients with malignant melanoma and then reintroduced into the patient, could be used to activate the patient's immune response.

• The use of liposome-bound plasmid DNA containing foreign histocompatibility genes to transduce tumor cells to enhance the immune response has also been proposed as a possible form of gene therapy in cancer.

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Introduce genes that selectively damage cancer cells

• Introduction of TNF or suicide genes like thymidine kinase gene of herpes virus to tumor cells.

• TK phosphorylates ganciclovir(prodrug) and causes DNA replication stall and resulting in the death of the cancer cells as well as surrounding cells through a 'bystander' effect.

• 'antiangiogenic' genes An example, is the inhibition of the angiogenic vascular endothelial growth factor (VEGF).

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Coronary artery disease

• VEGF is also a target for gene therapy in coronary artery disease where patients cannot be treated by angioplasty or coronary artery by-pass grafting. The aim is to overexpress VEGF and increase angiogenesis.

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Peripheral vascular disease

• introduction of genes which lead to proliferation of new vessels (e.g. VEGF) and the production of anticlotting agents.

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Autoimmune disorders

• Interleukin-1 (IL-1) is an immune system signal which triggers inflammation. For example, it has been proposed that gene therapy could be used to introduce genes for the IL-1 receptor antagonist protein into synovial cells in persons with rheumatoid arthritis, in which the inflammatory process is believed to play a key etiological role.

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