1. Severe combined immunodeficiencies (SCID) SCID consists of a group of genetic disorders characterized by a block in T lymphocyte differentiation that is variably associated with abnormal development of other lymphocyte lineages, i.e. B or NK lymphocytes or more rarely of the myeloid lineage. A genetic disorder in which both "arms" (B cells and T cells) of the adaptive immune system are impaired due to a defect in one of several possible genes. SCID is a severe form of heritable immunodeficiency. The overall frequency is estimated to 1 in 75 000±100 000 births

3. The proliferation, differentiation and TCR gene re-arrangement processes that occur in the thymus are tightly regulated. Any gene mutation leading to a blockage in the development of T cells in the thymus results in a deficiency, or sometimes even a complete absence, of mature T lymphocytes in peripheral blood and secondary lymphoid organs. Patients with such a deficiency cannot mount an effective immune response.

5. X-linked severe combined immunodeficiency Most cases of SCID are due to mutations in the gene encoding the common gamma chain (γc), a protein that is shared by the receptors for interleukins IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These interleukins and their receptors are involved in the development and differentiation of T and NK cells. Because the common gamma chain is shared by many interleukin receptors, mutations that result in a non-functional common gamma chain cause widespread defects in interleukin signalling. The result is a near complete failure of the immune system to develop and function, with low or absent T cells and NK cells. The common gamma chain is encoded by the gene IL-2 receptor gamma, or IL-2Rγ, which is located on the X-chromosome. For this reason, immunodeficiency caused by mutations in IL-2Rγ is known as X-linked severe combined immunodeficiency (T - NK - B + ).

6. Adenosine deaminase deficiency The second most common form of SCID after X-SCID is caused by a defective enzyme, adenosine deaminase (ADA), necessary for the breakdown of purines. ADA catalyses the deamination of adenosine (A) to inosine (I). The disease is caused by a mutation in a gene on chromosome 20. The gene codes for the enzyme adenosine deaminase (ADA). Without this enzyme, the body is unable to break down a toxic substance called deoxyadenosine. The toxin builds up and destroys infection-fighting immune cells called T and B lymphocytes

8. The effectiveness of the immune system depends upon lymphocyte proliferation. ADA deficiency leads to a T − B − NK − SCID caused by the accumulation of purine metabolites that are particularly toxic to more mature lymphocytes.

9. Omenn syndrome The manufacture of immunoglobulins requires recombinase enzymes derived from the recombination activating genes RAG-1 and RAG-2. These enzymes are involved in the first stage of V(D)J recombination, the process by which segments of a B cell or T cell's DNA are rearranged to create a new T cell receptor or B cell receptor (and, in the B cell's case, the template for antibodies). Certain mutations of the RAG-1 or RAG-2 genes prevent V(D)J recombination, causing SCID. Mutations in at least four genes coding for proteins involved in immunoglobulin- and TCR-rearrangement processes (RAG1, RAG2, Artemis and DNA Ligase IV) have been identified, all leading to a severe reduction of T and B cells (T − B − NK + SCID).

12. JAK3 Janus kinase-3 (JAK3) is an enzyme that mediates transduction downstream of the γ c signal. Mutation of its gene also causes SCID JAK-3 is a tyrosine kinase that is bound to the intracellular tail of gama chain and is activated upon cytokine binding to the multi chain receptor. JAK-3 phosphorylates STAT-5 protein. Phosphorylated STAT proteins dimerize and are translocated to the nucleus where they act as transcription inducing factors for several genes involved in progression of cell division Mutations in the genes encoding for the common gamma chain and its associated tyrosine kinase JAK3 lead to a T − B + NK − phenotype.

14. SCID and gene therapy

15. Cystic fibrosis Cystic fibrosis is an autosomal genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine. It is characterized by abnormal transport of chloride and sodium across epithelium, leading to thick, viscous secretions. CF is caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, is a deletion (Δ) of three nucleotides that results in a loss of the amino acid phenylalanine (F) at the 508th position on the protein.

16. This gene is required to regulate the components of sweat, digestive juices, and mucus. Although most people without CF have two working copies of the CFTR gene, only one is needed to prevent cystic fibrosis. CF develops when neither gene works normally However, there is no treatment for correcting the dysfunction of the chloride channel; existing therapies only alleviate the symptoms. Gene therapy aim to cure some of the effects of cystic fibrosis. Gene therapy aims to introduce normal CFTR to airway.

17. The strengths of gene therapy for CF are: a) it is a monogenic disease (CFTR mutation), b) the organ most affected is the lung, where the cells can be easily accessed, There are some problems associated with these methods involving efficiency (liposomes insufficient protein) and delivery (virus provokes an immune response).

18. AAV has emerged as a potential vector in gene therapy for CF because of a number of theoretical advantages: a)AAV has natural tropism for airway epithelial cells; b) AAV elicits little or no inflammatory response; c) AAV generally results in stable expression; d) Site-specific integration of AAV does not activate the possible oncogenes and the inserted gene can be maintained for a relatively long time in the host cell genome and stably expressed in vivo

20. Familial hypercholesterolemia Familial hypercholesterolemia (FH) is a genetic disorder characterized by high cholesterol levels, specifically very high levels of low-density lipoprotein (LDL, "bad cholesterol"), in the blood and early cardiovascular disease. Many patients have mutations in the LDLR gene that encodes the LDL receptor protein, which normally removes LDL from the circulation, or apolipoprotein B (ApoB), which is the part of LDL that binds with the receptor; mutations in other genes are rare.

22. Patients who have one abnormal copy (are heterozygous) of the LDLR gene may have premature cardiovascular disease at the age of 30 to 40. Having two abnormal copies (being homozygous) may cause severe cardiovascular disease in childhood. Heterozygous FH is a common genetic disorder, occurring in 1:500 people in most countries; homozygous FH is much rarer, occurring in 1 in a million births

23. The highest proportion of exon variants occurs in the ligand binding domain (exons 2-6)

24. Heterozygous FH is normally treated with statins, or other hypolipidemic agents that lower cholesterol levels. Homozygous FH often does not respond to medical therapy and may require other treatments The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved

25. Methods of gene delivery 1.Ex vivo gene transfer Involving isolation of cells from the patient, their in vitro genetic modification and selection followed by reimplantation of the transduced cells. The advantage of the ex vivo approach is that the transfection conditions can be carefully controlled and optimised and individual clones with the most desirable characteristics can be isolated to eliminate unmodified cells or cells with deleterious mutations before re-implantation. The disadvantages of the ex vivo approach are failure of cell engraftment and difficulties in returning the cells to the patient.

26. 2. In vivo gene transfer The vector is delivered directly to the organ. The gene transfer vector is injected into the bloodstream (systemic delivery) aiming at somatic cell delivery only or by use of specific cell targeting, preferentially to the tissues of interest (targeted delivery). The in vivo approach eliminates the need for engraftment after re-implantation and is therefore easier to perform, more cost effective and may be more applicable for use in countries with limited laboratory resources. Disadvantages of in vivo gene transfer are vector dilution, non- targeted, random, potentially genotoxic insertion into the host genome.

28. AAV mediated gene therapy for FH Vectors based on adeno-associated virus (AAV), a small (20-25 nm) non-enveloped DNA virus that is non-pathogenic, have a number of attributes that make them suitable for gene transfer to the liver for the treatment of FH. A single administration of recombinant AAV (rAAV) into the liver results in long-term transgenic protein expression without toxicity in a variety of animal models.

29. Reversal of hypercholesterolaemia was demonstrated in LDLR- /- mice fed with a high cholesterol diet after intraportal vascular injection of 1 × 10 12 AAV-2 vector particles encoding the murine LDLR driven by the CMV enhanced chicken b-actin promoter. Serum cholesterol progressively declined after vector administration and by 6 months, the aortic atherosclerotic lesion area was reduced 33% compared with control mice injected with saline.