Gene Editing Technologies, Ethics, and Applications

Gene Editing: Future Directions and Current Challenges

Addressing AAV Size Limitations for Cas9 Delivery

  • Problem: The gene encoding Cas9 is too large to fit into adeno-associated viruses (AAVs), which are commonly used for gene delivery.

  • Solutions:

    • Engineering smaller Cas9 variants: Some researchers are working to create smaller versions of the Cas9 protein.
    • Split Cas9 delivery: A more radical approach involves delivering the Cas9 protein in two halves that reassemble inside the cell. This is enabled by inteins which are protein introns.
Intein-Mediated Protein Splicing

  • Inteins are protein modules that can excise themselves from a precursor protein.

  • Mechanism:

    • Inteins juxtapose specific amino acid side chains.
    • This induces a chemical reaction that results in the intein being spliced out.
    • The two flanking protein fragments are joined together via a thioester bond, forming a continuous protein.
    • The formula for this process would be something like: Protein<em>N−Intein−Protein</em>C→Protein<em>N−S−Protein</em>C+InteinProtein<em>{N}-Intein-Protein</em>{C} \rightarrow Protein<em>{N}-S-Protein</em>{C} + Intein

  • Application to Cas9:

    • Cas9 is split into two halves.
    • An intein is attached to the N-terminus of the first half and another to the C-terminus of the second half.
    • The two halves are co-expressed and co-localized within the cell.
    • The inteins remove themselves, leaving the ends of the Cas9 halves ready to join.
    • The halves fuse to form a complete, functional Cas9 protein.
AAV-Mediated Delivery of Split Cas9

  • This approach utilizes two AAV vectors:

    • AAV Vector 1: Encodes the guide RNA and half of Cas9 (residues 2-573) with a nuclear localization sequence (NLS) and an N-terminal intein module.

      • AAV<em>1=gRNA+Promoter+1/2Cas9</em>(2−573)−NLS−NInteinAAV<em>{1} = gRNA + Promoter + 1/2Cas9</em>{(2-573)}-NLS-N_{Intein}

    • AAV Vector 2: Encodes the guide RNA and the other half of Cas9 (residues 574-1368) with an NLS and a C-terminal intein module.

      • AAV<em>2=gRNA+Promoter+1/2Cas9</em>(574−1368)−NLS−CInteinAAV<em>{2} = gRNA + Promoter + 1/2Cas9</em>{(574-1368)}-NLS-C_{Intein}

  • Both viruses transfect cells, and the Cas9 halves are expressed and localized to the nucleus.

  • The intein reaction occurs, causing the Cas9 halves to fuse and form a complete Cas9 protein, which then complexes with the guide RNA in order to create a functional gene editing system.

  • Experimentation: This system has been tested in pig and human models of muscular dystrophy.

Application to Muscular Dystrophy

  • Muscular Dystrophy Cause: Mutations in the dystrophin gene lead to an out-of-frame section, resulting in no functional dystrophin protein being produced and causing muscle wasting.

  • Solution: Instead of correcting the mutation, the approach involves excising an entire exon (e.g., exon 51).

  • This can be achieved through non-homologous end joining (NHEJ) after making cuts on either side of the exon.

  • Outcome: The resulting messenger RNA will lack the exon, and the protein will be shorter; however, it will still be functional.

  • Experimental Results (2020):

    • The split Cas9 system delivered via AAV vectors was used to excise exon 51.
    • This resulted in a shortened but functional dystrophin protein.
    • Improved muscle function, increased mobility, and prolonged lifespan were observed in the models.

Ethical Considerations of Germline Editing

  • Germline editing involves making modifications to a fertilized egg or early embryo, leading to changes in the resulting human being.

  • Ethical Concerns:

    • Potential pressures to make unnecessary changes to create "improved" children.
    • The need to restrict germline editing to cases where it is essential for the individual's health.
He Jiankui and the CRISPR Babies

  • He Jiankui, a researcher from China, famously created gene-edited babies (Lulu and Nana) to protect them against HIV.

  • Procedure:

    • IVF was performed, and the CCR5 gene was modified using CRISPR-Cas9 in the embryos.
    • The goal was to disable the CCR5 gene, thus preventing HIV from entering cells.

  • Ethical Issues:

    • The procedure was performed without proper ethical approvals.
    • It was deemed unnecessary for the children's health, as they did not have any intrinsic genetic problems.

  • Consequences for He Jiankui:

    • He was censored, fired from his university, and sentenced to three years in jail.
Global Regulations on Germline Editing
  • Gene editing of human embryos is banned in many countries, either through legislation or guidelines.

Summary of Gene Editing Technologies

  • Gene editing technologies have rapidly evolved, from zinc finger nucleases and TALENs to CRISPR-Cas systems.

  • These technologies have great potential for knocking out genes and making corrections to genes.

  • Ex vivo applications are working well and are being used in the clinic (in humans).

  • In vivo targeting is more challenging, but new approaches are promising.

  • Significant ethical and regulatory issues need to be addressed.

CRISPR-Cas9 Approach for Sickle Cell Anemia

Understanding Sickle Cell Anemia

  • Cause: A monogenic disorder caused by a single mutation in the beta-globin chain.

  • Mechanism: The mutation causes hemoglobin to polymerize within red blood cells, leading to a sickle shape.

  • Consequences:

    • Reduced oxygen transport
    • Clogging of arteries and veins
    • Symptoms include hemolysis, anemia, and ischemia
Fetal Hemoglobin and Sickle Cell Anemia

  • Hemoglobin tetramer contains two alpha-globin and two beta-globin subunits.

  • Fetal hemoglobin contains two alpha-globin and two gamma-globin subunits.

  • Persistence of Fetal Hemoglobin: Some individuals with the sickle cell gene are asymptomatic due to the continued expression of fetal hemoglobin (gamma form), leading to a benign phenotype.

  • This persistence can be caused by large deletions in the beta-globin gene cluster or point mutations in the promoters of the gamma-globin gene.

  • People with the beta-globin mutation and persistent fetal hemoglobin production have reduced polymer formation and are protected against sickle cell anemia.
    Morbidity∝1%Fetal HemoglobinMorbidity \propto \frac{1}{\%Fetal\ Hemoglobin}

Gene Editing Strategy to Turn on Fetal Hemoglobin

  • Why not correct the beta-globin mutation directly?

    • Mature red blood cells lack nuclei and DNA.
    • Precursor erythroid cells (stem cells) lack the machinery for homologous recombination.

  • Insight: Gamma-globin gene expression is repressed by the transcription factor BCL11A.

  • Targeting BCL11A:

    • Knocking out BCL11A would lead to increased gamma-globin expression and protection against sickle cell anemia.
    • However, BCL11A is an important transcription factor with regulatory roles in neuron development and B cells.

  • Solution: Regulate BCL11A specifically in erythroid cells.

  • Delete the BCL11A erythroid enhancer using genome editing, leading to a lack of BCL11A expression only in erythroid cells.

Mechanism of Action

  • In sickle cell disease, BCL11A represses gamma-globin, and the beta-globin mutation leads to sickle cell anemia.

  • Targeting the erythroid-specific enhancer of BCL11A by non-homologous end joining represses BCL11A, leading to sufficient gamma-globin production and healthy red blood cells.

  • Clinical Trials: This approach is being used in clinical trials in 2019.

Clinical Trial Results (2021)

  • Patients with sickle cell anemia or beta-thalassemia were treated with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer.

  • High percentage of alleles successfully modified.

  • No evidence of off-target effects.

  • After more than a year, patients had:

    • High levels of successful edits
    • Increased fetal hemoglobin
    • Elimination of blockages due to the diseases
In Vivo Targeting

  • More difficult than ex vivo approaches because of the need to deliver large tracts of DNA to the targets.

  • AAV vectors often too small for this purpose.

CCR5 as an HIV Therapy

  • Individuals with a mutated CCR5 receptor are not susceptible to HIV infection.

  • CCR5 is a co-receptor required for HIV entry into cells.

  • Destroying or eliminating CCR5 can protect cells from HIV infection.

Zinc Finger Nuclease Approach

  • As early as 2008, researchers used zinc finger nucleases to target and disrupt the CCR5 gene in the cell.

  • Zinc finger nucleases create double-stranded breaks in the DNA, and non-homologous end joining results in insertions or deletions that disrupt the gene.

  • The diagram shows the design of the zinc finger nuclease: 4\ Zinc\Fingers + Fok1\ Nuclease\ Monomer + 4\ Zinc\Fingers . They were specific for the targeted site.

  • Gene-edited cells had a significantly lower viral load compared to control cells when challenged with HIV.

Human Trials with Zinc Finger Nucleases

  • In 2014, 12 HIV-infected individuals on antiretroviral drugs had their T cells collected, cultured, and transfected with a zinc finger nuclease targeting CCR5.

  • The modified T cells were then transfused back into the patients.

  • All participants had elevated levels of T cells in their blood.

  • Six participants were able to stop their antiretrovirals, and HIV came back much more slowly than normal.

  • T cell levels remained high for weeks.

TALEN Gene Editing for Leukemia

  • Layla Richards, a young girl with leukemia, was treated with gene-edited CAR T cells after other therapies failed.

  • CAR T cells are T cells that have been given extra receptors to help them recognize cancer cells.

  • The researchers used a TALEN gene-edited approach to give the T cells the ability to not be recognized by the therapy against the CD52 receptor and also make sure that they would be matched inside the child.

  • The T cells were from a donor and were modified in culture using TALEN-mediated gene editing and then transfused into Layla.

  • This experimental treatment had amazing results and saved the little girl.

CRISPR-Cas9 System

  • Emmanuelle Charpentier and Jennifer Doudna recognized that the guide RNA could be fused with the trace RNA to make a system that could be used for gene editing.

  • Feng Zhang also developed CRISPR-Cas9 systems for gene editing and pioneered methods for using them in cells.

  • Both groups have been in competition, and there have been difficulties in working out the patents.

Somatic Cell Editing vs. Germline Editing

  • Somatic cell editing involves corrections that are not heritable to the next generations, whereas Germline editing does.

  • Somatic cell editing is used to treat an individual for their current disease.

Ex Vivo vs. In Vivo Approaches

  • Ex Vivo: Cells that need correcting can be removed from the body, cultured, corrected, and transplanted back into the original patient.

  • In Vivo: Systemic delivery of the components of the nuclease gene editing machinery into the bloodstream. Ideally with a targeted delivery into the particular organ of the cell of the body that's desired.

Considerations for Gene Editing

  • The gene editing is very limited in how long it persists in the body, if cell involved is short lived.

  • Homology directed repair only takes place in a cell that's undergoing cell cycle, conversely, non dividing cells can only use non homologous end joining.

  • The change must confer an advantage to the fitness of the cell, than the gene editing can really work well.

Nuclease Approaches in Human Therapeutics

  • Zinc finger nucleases, TALENs, and CRISPR-Cas9 have all been used in efforts to develop human therapeutics.
Factors to Consider

  • Recognition site lengths

  • Targeting constraints

  • Ease of engineering

  • Potential immunogenicity

  • Ease of delivery