Principles of therapy of hereditary diseases

Gene therapy options for a number of diseases are currently being explored.

Germ gene therapy

 * intervention in the gamete, zygote or embryonic cells at a very early stage of development
 * genetic change is found in all cells of the newly formed organism
 * The influence of bb, from which gametes are formed , is therefore transmissible to offspring
 * we cannot estimate the results in future generations of, ethical barriers before performing germline gene therapy

Somatic gene therapy

 * performing a genetic change in somatic calls, or tissues
 * tissues are selected according to the type of disease
 * manipulation in cells can be performed ex vivo, in vivo
 * Ex vivo = cells are harvested into a suitable environment and returned after therapy
 * In vivo = suitable for bb, which is impossible to cultivate or return to the body


 * the vector carrying the gene could be inserted directly into the tissue
 * the success of these experiments is very low
 * most suitable bb for gene therapy -long life, proliferate, can be easily obtained
 * Bone marrow stem cellsare an example - but they are poorly insulated

Somatic cell modification
Several approaches:


 * introducing a functional copy  of the gene into bb, the mutant gene remains unchanged
 * repairing the mutant gene or placing a working copy of the gene in place of the mutant gene
 * targeted inhibition of gene expression
 * targeted destruction of specific cells (significant in tumors)
 * destruction of specific cells of  the immune system

The goal is long-term expression of the introduced gene.

Thus, the foreign gene must integrate into the chromosome of the host cell, and bb must have the ability to further divide. The foreign gene is then transferred to the daughter cells - the gene integrates differently, o it is located in other places in subsequent cycles.

Viral vectors

 * viruses have developed efficient systems for inserting their genomes into human bb
 * the virus must be modified so that its genome does not harm the human cell
 * most of the  viral genome is deleted, replaced with human promoter and  regulatory regions
 * high efficiency

Retroviral vectors

 * the genome of retroviruses  jonsists of RNA, contains 3 genes (gag, pol, env) and the sequence phi, which is recognized by viral proteins - assembly of the viral particle
 * they have their own reverse transcriptase
 * upon entry into the host cell during division (membranes are disrupted)
 * cDNA attaches to host information při dělení (jsou porušené membrány)
 * the cloning capacity is 8 kb
 * The human gene vector is introduced into special cells that make many copies of human sequence retroviruses
 * modified retroviruses are then incubated with the patient's somatic cells (lymphocytes)
 * the human gene is inserted into the DNA of the host cells with high efficiency
 * disease : severe combined immunodeficiency (SCID)

Adenoviral vectors

 * dsDNA remains in the nucleus of a human cell, but does not integrate into its genome
 * the vector must be modified as well as the retrovirus
 * infect bb that do not divide (respiratory system)
 * cystic fibrosis – therapy - human CFTR gene in adenovirus - modified virus was applied to the epithelial airway in the form of an aerosol
 * low potency, only transient gene expression

Adeno-associated viral vectors

 * ssDNA, the replication of which depends on the presence of the virus
 * do not elicit any immune response
 * as adenoviruses can infect undivided se bb
 * hold a small insert – 5kb
 * factor IX vector for people with hemophilia B

Lentiviral vectors

 * complex retroviruses , can also infect non-dividing bb
 * holes in the nuclear envelope enter the nucleus
 * cloning sequence approx. 8 kb
 * antivirus = e.g. HIV

Problems associated with viral gene therapy

 * transient expression, low gene expression
 * difficult to  obtain specific bb, tissues (neurons)
 * the need for precise regulation of gene activity
 * potential danger of tumor transformation of the cell (accidental insertion of the virus into the cell may affect the expression of a minor gene, which may be a proto-oncogene)
 * immune response of the organism against the viral vector

Non-viral vectors

 * direct injection of DNA into tissue
 * firing of metal particles , that contain DNA
 * the association of DNA  with a molecule that is bound by receptors to the cell surface is followed by endocytosis
 * very low efficiency  in these methods

Liposomes

 * artificially formed phospholipid bilayer , particles that can hold a relatively large DNA insert
 * they can fuse with the cell, transferring the DNA insert into the cytoplasm
 * they do not contain peptides = they do not elicit an  immune response

Blockade of gene expression

 * gene products function in complexes of molecules (dimers)
 * the mutated protein  in the complex may affect their function
 * inhibition of transcription does not result in gene expression
 * replacement of gene damage by  homologous recombination

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 * 1) Gene therapy
 * 2) *eg. transformation by cloned genes
 * 3) **disease: adenosine deaminase deficiency (severe combined immunodeficiency )
 * 4) Enzyme induction
 * 5) *eg barbiturates
 * 6) **disease: congenital non-hemolytic jaundice (see differential diagnosis of jaundice)
 * 7) Enzyme replacement
 * 8) *eg tissue transplantation
 * 9) **disease: mucopolysaccharidosis
 * 10) *eg enzyme substitution
 * 11) **disease: trypsin deficiency
 * 12) Protein substitution
 * 13) *eg antihemophilic globulin
 * 14) **disease: hemophilia
 * 15) Vitamin substitution
 * 16) *eg. vitamin D
 * 17) **disease: vitamin D resistant rickets
 * 18) Product substitution
 * 19) *ex cortisone
 * 20) **disease: adrenogenital syndrome
 * 21) *eg thyroxine
 * 22) **disease: congenital hypothyroidism
 * 23) Substrate restriction in the diet
 * 24) *eg. AMK – Phenylalanin
 * 25) **disease: phenylketonuria (see Poruchy metabolismu aromatických a větvených aminokyselin)
 * 26) *eg sugars - galactose
 * 27) **disease: galactosemia
 * 28) *eg fats - cholesterol
 * 29) **disease: hypercholesterolemia (see Disorders of aromatic and branched chain amino acid metabolism ))
 * 30) Drugs that reduce the excess product of defective metabolism
 * 31) *eg. cholestyramin
 * 32) **disease: hypercholesterolemia (obsolete in the era of statins)
 * 33) *eg. penicilamin
 * 34) **disease: M.Wilson
 * 35) Replacement of the institution
 * 36) *eg kidney transplantation
 * 37) **DISEASE: polycystic kidney disease
 * 38) Removal of organ
 * 39) *eg colectomy
 * 40) **disease: familial colon polyposis

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