Lecture 24: Treatment of genetic diseases
Learning outcomes:
Understand the general approaches to the treatment of genetic diseases
Genetic diseases can be treated at any level from the mutant gene to the clinical phenotype.
Corrected mutated gene
Replace defective product
Improve defective protein’s function
Minimize consequences of defective protein’s absence
The strategy depends on the nature of the pathogenic allele and its clinical consequences
Challenges of treating genetic disease
Gene not identified of pathogenesis not well understood.
Prediagnostic fetal damage: Some variants act early in development or cause irreversible pathological changes before they are diagnosed.
Severe phenotypes are less amenable to intervention.
Specific variants may pose challenges
Some tissues are easier to target than others
Must minimize negative impacts resulting with treatment
Long-term assessment is critical
Understand the principle cofactor supplementation to improve the function of mutant enzymes as applied to variant PKU
The biochemical abnormalities of a number of inherited metabolic diseases may respond, sometimes dramatically, to the administration of large amounts of the vitamin cofactor of the enzyme impaired by the pathogenic variant.
Vitamin responsive inborn errors are among the most successfully treated of all genetic diseases.
Understand the general principle of ERT as applied to Gaucher Disease type I
Enzyme replacement therapy (ERT) is now established therapy for many lysosomal storage diseases.
Type 1 Gaucher disease was the first lysosomal storage disorder for which ERT was shown to be effective.
It is the most prevalent lysosomal storage disorder.
Autosomal recessive
Results from mutation in GBA1 gene causing β-glucocerebrosidase.
Loss of this hydrolase activity leads to accumulation of its substrate in the lysosome, which leads to anemia, gross enlargement of the liver and spleen, fatigue, and bone issues.
Extracellular administration of the intracellular enzyme caused dramatic benefits.
Effective at reducing Gaucher disease symptoms
Resolution of anemia and normalization of platelet counts
Weekly intravenous infusions increased hemoglobin levels
Understand the general principle of antisense oligonucleotide (ASO) therapy as applied to Duchenne Muscular Dystrophy
DMD is an X-linked recessive disorder
Mutation in gene encoding dystrophin
Complete frameshift mutation—everything downstream is disrupted
Dystrophin is important for skeletal muscles
Complete loss of function of dystrophin leads to loss of muscle tissue
Progressive muscle degeneration and weakness until death
Becker Muscular dystrophy (BMD)
Less severe phenotype
Reduced quantifies of dystrophin
Reading frame is not disrupted, only a region of the gene sequence is deleted
For DMD, if you could somehow get the reading frame back to normal, you could treat it.
Exon skipping refers to the use of molecular interventions to exclude an exon from a pre-mRNA that encodes a reading frame-disrupting variant, thereby reducing expression of the mutant gene.
If the number of nucleotides in the excluded exon is a multiple of 3, no frame shift will occur and, if the resulting polypeptide with the deleted amino acid retains sufficient function, a therapeutic result will benefit.
The most widely studied method of inducing exon skipping is through the use of ASOs, which are synthetic 15-35 nucleotide single stranded molecules that can hybridize to specific corresponding sequences in a pre-mRNA.
The goal of exon skipping in DMD is to convert a DMD pathogenic variant to an in-frame counterpart that generations a function dystrophin, just as the deletions that allow the production of a partially functioning dystrophin are associated with the milder phenotype of BMD.
Many DMD deletions span exon 50. Deletion of exon 50 created an out-of-frame transcript with a stop in exon 51.
But, deletion of exon 51 would restore the dystrophin reading frame enough to have a phenotype similar to BMD.
Understand the general principle of gene therapy, including the requirements and limitations
Gene therapy is the introduction of a biologically active gene into a cell to achieve a therapeutic benefit.
Useful for loss of function disorders
Requirements:
Molecular defect and mechanism fully known
Functional copy of the gene available
Appropriate vector (adenovirus, retrovirus, etc.)
Tight regulation of transferred gene
Appropriate target cell
Strong evidence of efficacy and safety
Regulator approval
Risks
Reaction to the vector
Insertional mutagenesis causing cancer
Insertional mutagenesis causing inactivation of essential gene
Be able to describe recent approaches for treating sickle cell disease by gene editing using CRISPR-Cas9
CRISPR-Cas9 uses engineered endonucleases containing a DNA-binding domain that will recognize a specific sequence in the genome, such as the sequence in which a missense variant is embedded.
Subsequently, a nuclease domain creates a DSB, and cellular mechanisms for homology-directed repair (HDR) then repair the break, introducing the wild-type nucleotide to replace the mutant one.
Sickle Cell disease is an autosomal recessive disorder caused by mutations in HBB, the gene encoding β-globin
Very common in African Americans
Symptoms include chronic hemolytic anemia, organ damage, pain, loss of vision, and shortened life span.
Fetal hemoglobin has 2 α subunits and two γ subunits
Adult hemoglobin has 2 α subunits and two β subunits
In sickle cell, there are mutations in the β subunits, so the fetal form is functional, but when it switches from fetal to adult, that’s where the issue is.
BCL11A is a zinc-finger transcription factor that represses γ-globin when switching from fetal to adult.
Deletion of an enhancer in BCL11A with CRISPR can stop the transition from γ to β, therefore keeping the functional γ globin.
Type I Gaucher Disease
Becker Muscular Dystrophy (BMD)
Enzyme replacement therapy (ERT)
Antisense oligonucleotides (ASOs)
Exon-skipping therapy
Gene therapy
Gene therapy vector
Gene therapy target cell
BCL11A
Fetal hemoglobin
Adult hemoglobin