• Information
  1. Potential Gene Therapy Targets
  2. Gene Delivery Systems
  3. Correction of Recessive Genetic Mutations
  4. Correction of Dominant Genetic Mutations
  5. Gene Therapy of Cancer
  6. Muscle Mediated Gene Therapy
  7. Cystc Fibrosis
  8. Cardiovascular Disease
  9. Gene-Based Vaccines

A. Potential Gene Therapy Targets

1. Replacement of "Missing" or "Null" Genes
2. Replacement of Abnormal Genes (whose products are expressed)
3. Gene Therapy of Neoplastic Diseases
4. Increase Expression of Desired / Protective Genes
   1. Drug resistance glycoproteins (such as mdr1/PGP1) in normal cells [3]
   2. Anti-inflammatory proteins in diseased arthritic joints
5. Gene Therapy of AIDS
6. Treatment of autoimmune disease
7. Gene-based Vaccination [5]

B. Gene Delivery Systems [1,2,3]

1. Naked DNA
2. Liposomes / Cationic Lipid Mediated Transfer [7]
3. Viral vectors are becoming less attractive due to side effect issues
4. Retrovirus
   1. Requires packaging cell line expressing gag, pol and env
   2. Recipient cells must be proliferating in order to integrate new gene
   3. Over 300 patients have received retroviral vector-genes with no adverse effects
   4. Successful prolonged gene expression after ex vivo transfer has been achieved [6]
   5. Gammaretroviral vector has been used to correct SCID in humans [4,6]
5. Adenovirus
6. Adeno-Associated Virus (AAV)
7. Herpes Simplex Virus (HSV) Type 1
8. Adenovirus Coat Protein
9. Delivery may be ex vivo or in vivo
10. Major problem in all areas of gene therapy is immune responses to vectors

C. Correction of Recessive Genetic Mutations

1. These are theoretically the easiest to correct
2. Most of these disorders are monogenic recessive
3. Even if abnormal gene is expressed, it does not interfere with normal protein function
4. Normal (replacement) gene needs to be transferred into cell and expressed
5. No specific insertion site (into genome) will be required
6. Examples
   1. Severe combined immunodeficiency (SCID): autosomal and X-linked forms
   2. X-Linked SCID: defects in gene encoding common cytokine receptor gamma chain [4]
   3. Autosomal SCID: adenosine deaminase deficiency
   4. Factor VIII deficiency
   5. Hypercholesterolemia
7. Other immunodeficiency diseases are being evaluated
8. Specific Gene Regulation
   1. Many genes can be expressed at ectopic sites and will function, improve symptoms
   2. However, some genes will have to be expressed in appropriate tissues at a given time
   3. ß-globin expression, abnormal thalassemia, requires cell and temporal specificity
   4. Cystic fibrosis gene transfer (CFTR) is under intensive study [7,8]
   5. alpha1-antitrypsin deficiency (panacinar emphysema) is also being studied [8]
9. Correction of X-Linked SCID [4,6]
   1. Ex vivo gene transfer with defective retroviral vector achieved
   2. Common gamma chain gene expression >2.5 years after transfected CD34+ cells infused
   3. Thymopoiesis, T and B lymphocytes documented
   4. Peripheral T cell show T cell receptor diversity
   5. B-cells matured and produced increasing rates of somatically mutated immunoglobulin (Ig)
   6. Serum Ig levels restored to level sufficient to avoid IV Ig treatments in some patients
   7. Vaccination stimulated antibody production

D. Correction of Dominant Genetic Mutations

1. Abnormal gene products are expressed and may interfere with normal gene products
   1. If there is interference with normal function, the disorder is usually dominant
   2. If there is no interference with normal function, the disorder is recessive
2. For dominant mutations, actual replacement of the abnormal gene may be required
3. Specific disruption of abnormal gene by transferred normal gene may be required
   1. Such targetted gene replacement therapies may be difficult
   2. The technology for this "homologous recombinantion" exists but is not efficient
4. Examples: sickle cell anemia, familial retinoblastoma (abnormal RB protein)
   1. In sickle cell anemia, the abnormal hemoglobin, HbSS, interferes with normal function
   2. If high enough normal Hb (HbA) can be achieved, then symptoms may be reduced
   3. Heteromeric Hb molecules should reduce symptoms

E. Gene Therapy of Cancer [6,8]

1. Introduction of drug susceptibility ("suicide") genes into cancer cells
   1. Introduction of normal p53 genes into tumor cells having abnormal p53
   2. This should enhance tumor response to radiation and/or chemotherapy
   3. Introduction of herpes thymidine kinase gene to tumors makes susceptible to ganciclovir
   4. HSVtk metabolism of ganciclovir leads to bystander killing by diffusion of toxic metabolites, thus potentially enhancing activity
2. Introduction of Immune enhancing molecules into the tumor cells
   1. Expression of major histocompatibility proteins (MHC)
   2. Increased MHC proteins on a subset of tumor cells may increase immunogenicity
   3. Expression of T cell coactivation molecules on the tumor cells
   4. This may allow tumor cells themselves to act as immune accessory cells
   5. Expression of cytokine genes in the tumor cells
3. Expression of specific cytokine genes in lymphocytes and other cells in cancer patients
4. Introduction of chemotherapy-protecting genes into normal cells
   1. Introduction of drug resistance (eg. mdr-1) genes into normal hematopoietic cells
   2. Use with autologous bone marrow transplantation during/after high dose chemotherapy
5. Genetic markers such as neo resistance gene can be used to follow specific cell homing
   1. This is useful in animal models of tumor cell metastates
   2. Has been empolyed to follow TIL and CTL cells in therapy of cancer patients
   3. Gene marking demonstrated tumor cell reinfusion in bone marrow transplant

F. Muscle Mediated Gene Therapy [9]

1. Muscle cells can be manipulated fairly easly for gene expression
   1. Muscle biopsy explants will give rise to myoblasts in culture
   2. Naked (eg. plasmid) DNA injected into muscle will be taken up and genes expressed
   3. Naked DNA injected into muscle may make ideal vaccination methods
2. These cells can be infected with retrovirus containing gene of interest
   1. Gene expression is very efficient in these cells
   2. Post-translational modifications are generally carried out correctly
3. Myoblasts expressing recombinant protein can be reinjected into human muscle [10]
   1. Injected myoblasts fuse into the host muscle fibers
   2. This process is fairly efficient, including gene expression from the fiber
   3. Proteins secreted from the fibers enter recipient's bloodstream

G. Cystc Fibrosis [7,11,12]

1. Mutations in the CFTR gene lead to malfunctioning transport protein
2. Most of the CFTR mutations appear to be recessive
3. Adenoviral vectors carrying normal CFTR cDNA have been used for transfer
4. Major problem at present is immune response to viral proteins expressed on infected cells
5. Modifications of the viral vectors is currently a major focus of research
6. Whether CFTR can be targetted with adenovirus to pancreas and other organs is not clear

H. Cardiovascular Disease [13]

1. Development of collateral blood vessels
   1. Vascular Endothelial Growth Factor (VEGF)
   2. Fibroblast Growth Factor ß (FGFß)
   3. Particularly for myocardial ischemia and peripheral vascular disease
2. Anti-Angiogenesis Therapy
   1. Blockade of restenosis (after angioplasty) and vein graft thickening
   2. Local delivery of a variety of agents is preferred

I. Gene-Based Vaccines [5]

1. DNA and other gene-based vaccines under development
2. Designed to generate both cellular and humoral (antibody) immune responses
3. Live versus naked DNA vector systems being investigated
4. Plasmid DNA vaccines are probably safe and stimulate good immunity
5. Mixed modality vaccines also under development

References

1. Ratko TA, Cummings JP, Blebea J, Matuszewski KA. 2003. Am J Med. 115(7):560
2. Hanania EG, Kavanagh J, Hortobagyi G, et al. 1995. Am J Med. 99(5):537
3. Brenner MK. 1996. NEJM. 335(5):340
4. Gaspar HB, Parsley KL, Howe S, et al. 2004. Lancet. 364(9452):2181
5. Srivastava IK and Liu MA. 2003. Ann Intern Med. 138(7):550
6. Hacein-Bey-Abina S, Le Deist F, Carlier F, et al. 2002. NEJM. 346(16):1185
7. Alton EWFW, Stern M, Farley R, et al. 1999. Lancet. 353:947
8. Albelda SM, Wiewrodt R, Zuckerman JB. 2000. Ann Intern Med. 132(8):649
9. Blau HM and Springer ML. 1995. NEJM. 333(23):1554
10. Mendell JR, Kissel JT, Amato AA, et al. 1995. NEJM. 333(13):832
11. Knowles MR, Hohneker KW, Zhou Z, et al. 1995. NEJM. 333(13):823
12. Schwiebert EM, Benos DJ, Fuller CM. 1998. Am J Med. 104(6):576
13. Yla-Herttuala S and Martin JF. 2000. Lancet. 355(9199):213