Gene therapy aims to "cure" or reduce the severity of genetic diseases.
Two main approaches:
Gene editing using CRISPR.
Inserting a functional allele of the gene into cells.
Examples:
Sickle-cell anemia treatment using CRISPR (approved).
Functional allele insertion treatments for hemophilia A (approved, using a viral vector).
Sickle-Cell Anemia (SCA)
SCA is caused by an autosomal recessive allele.
Missense mutation at codon 6 of the beta-globin gene (HBB).
Coding strand change: 5’ GAG 3’ to 5’ GTG 3’.
This mutation causes hemoglobin molecules to stick together noncovalently, forming long chains inside red blood cells (RBCs).
Sickle-Cell Anemia: Genetic Basis
Healthy Individuals:
Have two working HBB genes on chromosome 11.
Produce working beta-globin protein which combines with alpha-globin to form hemoglobin.
Result: Round, flexible RBCs that efficiently carry oxygen.
Individuals with Sickle-Cell Anemia:
Have two altered HBB genes on chromosome 11.
Produce altered beta-globin protein that stick to one another, forming long fibers.
Result: Stiff and misshapen RBCs that carry oxygen, but the fibers impair their function.
Monohybrid Cross
Illustrative example of a monohybrid cross with probabilities, presumably related to inheritance patterns (0.5 x 0.5 = 0.25).
Pedigree Analysis of Sickle-Cell Anemia
Analysis of a pedigree chart to determine genotypes of individuals (III-1 and III-2).
Determining the mode of inheritance by ruling out X-linked recessive based on affected individuals (IV-1).
Genotype and Allele Frequencies for SCA
Prevalence in African Americans:
Approximately 1 in 400 has sickle-cell anemia (cc genotype).
Approximately 1 in 10 is a carrier (Cc genotype).
Genotype Frequencies:
f(cc) = 0.0025 (1/400).
f(Cc) = 0.1.
f(CC) = 1 – (0.1 + 0.0025) = 0.8975.
Allele Frequencies:
f(C) = 0.8975 + (0.5 * 0.1) = 0.9475.
f(c) = 0.0025 + (0.5 * 0.1) = 0.0525.
Predicting Genotypic Frequencies
Using allele frequencies to predict genotypic frequencies in the population.
Hemoglobin Composition
Adult Hemoglobin:
In mammals, adult hemoglobin (α<em>2,β</em>2) is produced starting just before birth.
Fetal Hemoglobin:
During fetal development, a different version (α<em>2,γ</em>2) is produced.
Differential Gene Regulation and Hemoglobin
RBCs are produced by stem cells in the bone marrow.
Expression of different hemoglobin subunit genes is controlled by stem cells.
This results in RBCs containing either fetal (α<em>2,γ</em>2) or adult (α<em>2,β</em>2) hemoglobin.
BCL11A:
BCL11A is a repressor protein that works with a silencer element to shut off expression of the gamma ($\gamma$) gene.
CRISPR Gene Therapy Mechanism:
The CRISPR gene therapy removes a regulatory sequence needed for BCL11A expression.
Without BCL11A, stem cells produce RBCs containing (α<em>2,γ</em>2) hemoglobin, even after birth.
Exa-cel Mechanism of Action
BCL11A Role : BCL11A is a transcription factor that normally represses the expression of fetal hemoglobin genes in adult red blood cells.
The figure depicts how BCL11A, influenced by the GATA1 erythroid lineage-specific enhancer, affects globin gene expression.
Enhancer: It is located near the BCL11A gene on chromosome 2, plays a role in regulating BCL11A expression specifically in erythroid cells.
Without the functional BCL11A protein, the embryonic, fetal, adult hemoglobin will no longer be expressed.
Globin Gene Cluster: The cluster contains genes for different globin subunits (embryonic, fetal, and adult forms) which is located on Chromosome 11 which will be affected by the action of BCL11A.
LCR helps control the expression of these genes during development.
Exa-cel Manufacturing Process
Clinical Trial Site:
Stem cells are collected from the patient via mobilization and apheresis.
Myeloablative conditioning with busulfan is performed.
Backup cells are kept at the site as a safety measure.
Cryopreserved cells are received at the site and stored until infusion.
Central Manufacturing Location:
CD34+ cells are isolated.
Cells are electroporated to deliver CRISPR/Cas9 for gene editing (CTX001).
CRISPR/Cas9-modified stem cells (CTX001) are cryopreserved, tested, and released.
Back to Clinical Trial Site:
CTX001 cells are thawed and infused into the patient.
Monitoring for engraftment and immune reconstitution follows.
Mechanism of Gamma Globin Expression
CRISPR-edited bone marrow cells can no longer produce BCL11A protein.
Transcription of the gamma ($\gamma$) hemoglobin genes is reactivated.
Transcription of the mutant sickle hemoglobin ($\beta^S$) alleles stops.
Gene-edited stem cells now produce functional (α<em>2,γ</em>2) hemoglobin in the patient.
Outcomes of Exa-cel Treatment
Approved for use in the US in 2023.
Side effects are comparable to bone marrow transplants.
High cost: $2.2 million, making it unaffordable for most people.
Affordability and access are major ethical/moral issues for gene therapies.