The Treatment of Genetic The Treatment of Genetic Disease Disease

Lecture 6-a

The understanding of genetic disease at a molecular level is the foundation of rational therapy.

The objective in treating genetic disease is to eliminate or ameliorate the effects of the disorder, not only on the patient but also on his or her family.

THE CURRENT STATE OF TREATMENT OF GENETIC DISEASE

Genetically Complex Diseases

Environmental interventions, such as medications and lifestyle or diet changes, may have a greater impact on the management of genetically complex diseases.

For example, cigarette smoking is an environmental factor that all patients with age-related macular degeneration or emphysema should strictly avoid.

A striking example of a complex disorder for which standard medical therapy is increasingly successful is type 1 diabetes mellitus, in which intensive insulin replacement therapy greatly improves the outcome.

Surgical treatment of multifactorial disorders can also be highly successful. For example, three structural abnormalities (congenital heart defects, cleft lip and palate, and pyloric stenosis).

TREATMENT STRATEGIES

Therapy Directed at the Clinical Phenotype Treatment at the level of the clinical phenotype

includes all the medical or surgical interventions that are not unique to the management of genetic disease.

Include: – Avoidance– Dietary restriction– Replacement– Diversion– Inhibition– Depletion

Dietary Restriction Dietary restriction is one of the oldest and

most effective methods of managing genetic disease.

Diseases involving more than several dozen loci are currently managed in this way.

It usually requires lifelong compliance with a restricted and often artificial diet.

Many of the diseases treatable in this manner involve amino acid catabolic pathways, and therefore severe restriction of normal dietary protein is usually necessary.

ReplacementThe provision of essential metabolites,

cofactors, or hormones whose deficiency is due to a genetic disease is simple in concept and often simple in application.

E.g., congenital hypothyroidism, 10% to 15% of which is monogenic in origin. This disorder results from a variety of defects in the formation of the thyroid gland or of its major product, thyroxine.

Diversion

Diversion therapy is the enhanced use of alternative metabolic pathways to reduce the concentration of a harmful metabolite.

A successful application of diversion therapy is in the treatment of the urea cycle disorders.

If the cycle is disrupted by an enzyme defect such as ornithine transcarbamylase deficiency, the consequent hyperammonemia can be only partially controlled by dietary protein restriction.

The ammonia can be reduced to normal levels by diversion to metabolic pathways that are normally of minor significance, leading to synthesis of harmless compounds.

Thus, the administration of large quantities of sodium benzoate forces its ligation with glycine to form hippurate, which is excreted in the urine.

Glycine synthesis is thereby increased, and for each mole of glycine formed, one mole of ammonia is consumed.

Figure 13-4 The strategy of metabolite diversion. In this example, ammonia cannot be removed by the urea cycle because of a genetic defect of a urea cycle enzyme. The administration of sodium benzoate diverts ammonia to glycine synthesis, and the nitrogen moiety is subsequently excreted as hippurate.

A similar approach has been successful in reducing cholesterol level in heterozygotes for familial hypercholesterolemia.

By the diversion of an increased fraction of cholesterol to bile acid synthesis, the single normal low-density lipoprotein (LDL) receptor gene of these patients can be stimulated to produce more hepatic receptors for LDL-bound cholesterol

This treatment achieves significant reductions in plasma cholesterol because 70% of all LDL receptor-mediated uptake of cholesterol is by the liver.

The increase in bile acid synthesis is achieved by the oral administration of non-absorbable resins such as cholestyramine, which bind bile acids in the intestine and increase their fecal excretion.

This example illustrates clearly an important principle: autosomal dominant diseases may sometimes be treated by increasing the expression of the normal allele.

InhibitionThe pharmacological inhibition of enzymes

is sometimes used to modify the metabolic abnormalities of inborn errors.

Familial hypercholesterolemia also illustrates this principle. When the cholesterol load is decreased by diverting it to other compounds or by removing it with physical methods, the liver tries to compensate for the decreased cholesterol intake by up-regulating cholesterol synthesis.

Consequently, the treatment of familial hypercholesterolemia heterozygotes is more effective if hepatic cholesterol synthesis is simultaneously inhibited by a statin, a class of drugs that are powerful inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis.

High doses of a statin typically effect a 40% to 60% decrease in plasma LDL cholesterol levels in familial hypercholesterolemia heterozygotes; when a statin is used together with cholestyramine, the effect is synergistic, and even greater decreases can be achieved.

Figure 13-5 Rationale for the combined use of a bile acid-binding resin and an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) in the treatment of familial hypercholesterolemia heterozygotes.

DepletionGenetic diseases characterized by the

accumulation of a harmful compound are sometimes treated by direct removal of the compound from the body. This principle is exemplified by the use of phlebotomy to alleviate the iron accumulation that occurs in hemochromatosis.

Table 13-3. Treatment of Genetic Disease by Metabolic Manipulation

Type of Metabolic Intervention

Substance or Technique Disease

Avoidance Antimalarial drugs Glucose-6-phosphate dehydrogenase deficiency

Isoniazid Slow acetylators

Dietary restriction PhenylalanineGalactose

PhenylketonuriaGalactosemia

Replacement Thyroxine Congenital hypothyroidism

Biotin Biotinidase deficiency

Diversion Sodium benzoate Urea cycle disorders

Oral resins that bind bile acidsDrugs that block the intestinal absorption of cholesterol

Familial hypercholesterolemia heterozygotes

Inhibition Statins Familial hypercholesterolemia heterozygotes

Depletion LDL apheresis (direct removal of LDL from plasma)

Familial hypercholesterolemia homozygotes

THE MOLECULAR TREATMENT OF THE MOLECULAR TREATMENT OF DISEASEDISEASE

Treatment at the Level of the ProteinTreatment at the Level of the Protein

Enhancement of Mutant Protein Function with Small Molecule Therapy

Small molecules are that class of compounds with molecular weights in the few hundreds to thousands. Synthetic or natural.

Vitamins, non-peptide hormones, and most drugs are classified as small molecules.

Vitamin-Responsive Inborn Errors of Metabolism

The biochemical abnormalities of a number of metabolic diseases may respond, to the administration of large amounts of the vitamin cofactor of the enzyme impaired by the mutation.

In homocystinuria due to cystathionine synthase deficiency, e.g., about 50% of patients respond to the administration of high doses of pyridoxine (vitamin B6, the precursor of pyridoxal phosphate); in most of these patients, homocystine disappears from the plasma.

The increased pyridoxal phosphate concentrations may overcome reduced affinity of the mutant enzyme for the cofactor or stabilize the mutant enzyme.

Nonresponsive patients generally have no residual cystathionine synthase activity to augment.

Figure 13-7 The mechanism of the response of a mutant apoenzyme to the administration of its cofactor at high doses. Vitamin-responsive enzyme defects are often due to mutations that reduce the normal affinity (top) of the enzyme protein (apoenzyme) for the cofactor needed to activate it. In the presence of the high concentrations of the cofactor that result from the administration of up to 500 times the normal daily requirement, the mutant enzyme acquires a small amount of activity sufficient to restore biochemical normalcy.

Small Molecules to Increase the Folding of Mutant Polypeptides

Many mutations disrupt the ability of the mutant polypeptide to fold normally. If the folding defect could be overcome, the mutant protein would often be able to resume its normal activity.

The administration of small molecules may be used to overcome a folding defect.

Folding mutants of membrane proteins, for example, fail to pass normally through the endoplasmic reticulum and get "stuck" there, leading to their degradation.

E.g., the ΔF508 mutation of the cystic fibrosis protein. The mutant ΔF508 polypeptide is recognized by a calcium-dependent chaperone protein in the endoplasmic reticulum, retained there, and degraded.

An extraordinary correction of this defect has been obtained in mice carrying the ΔF508 mutation by the administration of curcumin, a nontoxic mixture of compounds derived from turmeric, a spice found in curry.

Curcumin inhibits a calcium pump in the endoplasmic reticulum, thereby impairing the binding of the mutant ΔF508 protein by the calcium-dependent chaperone.

The treated mice had a normalization of chloride transport in the gut and nasal epithelium and dramatically increased rates of survival.

Small Molecule Therapy to Allow Skipping over Mutant Stop Codons

Aminoglycoside antibiotics, encourage the translational apparatus to "skip over" a premature stop codon and instead to misincorporate an amino acid that has a codon comparable to that of the termination codon.

In this way, for example, Arg553Stop in CF is converted to 553Tyr, a substitution that generates a CFTR peptide with nearly normal properties.

Protein AugmentationThe prevention or arrest of bleeding

episodes in patients with hemophilia by the infusion of plasma fractions enriched for factor VIII is the prime example.

Augmentation of an Extracellular Protein: α1-Antitrypsin Deficiency

Human α1AT can be infused intravenously in doses sufficiently large to maintain the interstitial fluid α1AT concentration at an effective inhibitory level for 1 week or even longer.

An alternative approach still being studied involves the delivery of α1AT directly to the lungs by aerosol inhalation. This route of administration is more attractive, since it requires only 10% of the intravenous dose of α1AT.

Enzyme Replacement Therapy: Extracellular Enzyme Replacement Therapy: Extracellular Augmentation of an Augmentation of an IntracellularIntracellular Enzyme Enzyme

Adenosine Deaminase DeficiencyAdenosine Deaminase Deficiency

Figure 13-8 Adenosine deaminase (ADA) converts adenosine to inosine and deoxyadenosine to deoxyinosine. In ADA deficiency, deoxyadenosine accumulation in lymphocytes is lymphotoxic, killing the cells by impairing DNA replication and cell division to cause severe combined immunodeficiency (SCID).

Adenosine deaminase (ADA) is a critical enzyme of purine metabolism that catalyzes the deamination of adenosine to inosine and of deoxyadenosine to deoxyinosine.

The pathology of ADA deficiency, an autosomal recessive disease, results entirely from the accumulation of toxic purines, particularly deoxyadenosine, in lymphocytes severe combined immunodeficiency.

The current treatment of choice, however, is bone marrow transplantation from a fully HLA-compatible donor.

Administration of the bovine ADA enzyme has been shown to be effective.

Modified Adenosine DeaminaseThe infusion of bovine ADA modified by

the covalent attachment of an inert polymer, polyethylene glycol (PEG), has been found to be superior to the use of the unmodified ADA enzyme.

Enzyme Replacement Therapy: Targeted Augmentation of an Intracellular Enzyme

Enzyme replacement therapy (ERT) is now established therapy for two lysosomal storage diseases, Fabry disease and Gaucher disease.

Fabry disease: α-galactosidase deficiency– accumulation of globosides (glycosphingolipids)– Reddish purple skin rash– kidney and heart failure– burning pain in lower extremities

Gaucher Disease This autosomal recessive condition is due to a

deficiency of the enzyme glucocerebrosidase. Its substrate, glucocerebroside, is a complex lipid normally degraded in the lysosome.

The disease results from glucocerebroside accumulation, particularly in the lysosomes of macrophages in the reticuloendothelial system, leading to gross enlargement of the liver and spleen.

In addition, bone marrow is slowly replaced by lipid-laden macrophages ("Gaucher cells") that ultimately compromise the production of erythrocytes and platelets, producing anemia and thrombocytopenia. Bone lesions cause episodic pain, osteonecrosis, and substantial morbidity.