Week 8.2 CRISPR-Cas9: Biological Mechanisms, Therapeutic Applications, and Ethical Considerations

Introduction to CRISPR and Gene Therapy

• Defining the Scope: All topics discussed (viral vectors, CRISPR) fall under the umbrella of gene therapy, which is the modification of gene expression within the body.

• Distinction from Previous Lecture:

  • Dr. Lipschitz discussed gene therapy via viral vectors (using viruses to deliver therapeutic genes).

  • CRISPR represents gene therapy using a different molecular mechanism based on genome editing rather than simple supplementation.

• Lecture Structure:

  • Part 1: Basic biology of CRISPR.

  • Part 2: Somatic context of gene editing.

  • Part 3: Germline context of gene editing.

General Perspectives: Benefits and Concerns of CRISPR

• Potential Concerns and Critiques:

  • Neo-Eugenics: Based on readings by Syfyan and Garland-Thompson, there is concern about "editing out" genetic traits that are integral to human identity and variation.

  • Autonomy: Questions arise regarding the respect for the autonomy of future persons (embryos) and the limits of parental decision-making.

  • Designer Babies: Fear of creating "designer babies" (e.g., selecting for intelligence or strength) leading to social stratification between the genetically "enhanced" and "unenhanced."

  • Global Access: The high cost of treatment and lack of infrastructure may prevent these technologies from reaching developing countries where genetic conditions (like sickle cell) are most prevalent.

• Potential Benefits:

  • Symptom Relief: Real-world examples like Victoria Gray demonstrate major relief from debilitating symptoms of conditions like sickle cell anemia.

  • Site-Specific Precision: CRISPR allows for edits at the endogenous locus of a gene, reducing the risk of disrupting important functional genes (a common problem with viral vectors).

  • Technical Versatility: Emergent techniques like base editing allow for precise single-nucleotide swaps.

The Basic Biology of CRISPR-Cas9

• Discovery and Recognition:

  • Recognized as the "Breakthrough of the Year" by Science Magazine in 2015.

  • Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry in 2020 for their work on the system.

• Origins: It is a naturally occurring immune system in bacteria used to protect against viruses, which scientists modified for laboratory use.

• Molecular Components:

  • Target Gene: The specific section of DNA containing a variant to be modified.

  • Guide RNA ($gRNA$): A yellow hairpin-like structure designed to match the sequence of the target gene. It follows nucleic acid complementarity rules (e.g., $C$ binds with $G$, and $A$ binds with $U$ in RNA) and acts as a "finder" to direct the enzyme to the correct genomic location.

  • Cas9 Enzyme: Often described as "molecular scissors." It is an enzyme that catalyzes a double-stranded break in the DNA at the location specified by the $gRNA$.

• The Mechanism:

  • The complex (enzyme + $gRNA$) binds to the DNA.

  • A cut is induced.

  • The cell attempts to repair the break. This repair can either naturally disrupt a gene (gene knockout) or incorporates a new piece of DNA if provided (gene paste/knock-in).

  • Comparison to Older Tech: Older methods include Zinc Fingers and Talens, but CRISPR is simpler, more efficient, and easier to program.

Technical Challenges and Safety Risks

• Precision Limits: While CRISPR is far more precise than past tools, it is not perfect.

• Off-target Edits: The $gRNA$ may accidentally bind to a similar sequence elsewhere in the genome, causing unintended cuts and modifications.

• Unexpected On-target Effects: Even when the tool hits the right spot, the resulting repair may be unexpected, such as:

  • Large, unintended deletions of genetic material.

  • Chromosomal flipping or swapping of sequences on homologous chromosomes.

  • Unintended patterns of recombination.

• Ethical Alignment: Arguments regarding the harm caused by off-target effects align with the bioethical principle of Non-maleficence (the duty to avoid harm).

Somatic vs. Germline Gene Editing

• Somatic Gene Editing:

  • Target: Specific differentiated cells (e.g., blood cells, liver cells, neurons).

  • Impact: Restricted to the individual being treated.

  • Generational Transfer: Edits are not passed to offspring because they do not affect the germ cells (egg and sperm).

  • Regulatory Standing: Generally more well-accepted and regulated.

• Germline Gene Editing:

  • Target: Early embryos or gametes.

  • Impact: Every cell in the resulting organism will carry the edit.

  • Generational Transfer: The edit will be incorporated into the person's own germline and passed to their future children.

  • Regulatory Standing: Highly controversial and widely restricted due to the permanence of potential errors.

Case Study: Somatic CRISPR for Sickle Cell Anemia

• Condition: Sickle cell disease is caused by a genetic variant in the hemoglobin gene, leading to painful cell clogging and reduced oxygen transport.

• FDA Approval (2023): The first approved CRISPR therapy for sickle cell (Exa-cel/Casgevy) was authorized alongside a viral vector treatment (Lifygenia).

• The Mechanism (Ex Vivo):

  1. Red blood cells are removed from the patient.

  2. Cells are modified in a dish (Ex Vivo) using CRISPR.

  3. Modified cells are screened for the correct edits and the absence of off-target effects.

  4. Cells are re-infused into the patient.

• The Biological Workaround: Rather than fixing the mutated adult hemoglobin gene, the therapy targets the $HGF$ gene to turn back on Fetal Hemoglobin ($HbF$), which compensates for the defective adult hemoglobin.

• Economics and Accessibility:

  • Cost is estimated between $2 \times 10^6$ and $3 \times 10^6 \text{ USD}$ per treatment.

  • Questions remain regarding whether insurance will cover these costs and how these treatments can reach areas like Africa and Asia where sickle cell rates are highest.

• Bespoke Treatments: Current research explores "n-of-1" therapies for ultra-rare conditions (e.g., $CPS1$ liver enzyme deficiency), though funding and scaling these personalized treatments remains difficult.

The CRISPR Baby Scandal and Germline Ethics

• U.S. Policy: The FDA is currently prohibited by Congress from even considering applications for germline editing, creating an effective ban. The NIH will not fund germline research.

• 2017 Guidelines: The National Academy of Sciences released guidelines suggesting germline editing could be permissible only for serious diseases, when no alternatives exist, and using well-understood genes.

• He Jiankui (2018):

  • A Chinese scientist announced the birth of the first gene-edited babies (twins).

  • He targeted the $CCR5$ gene, which encodes a cell surface protein that HIV uses to enter cells. He aimed to provide HIV resistance.

  • Scientific Backlash: Criticized for lack of transparency, skipping preclinical data, and ethical lapses in the consent process.

  • Technical Failures: The children were found to have mosaicism (some cells edited, some not) and some were heterozygous for the change, meaning the intended resistance was not fully achieved.

  • Outcome: He Jiankui was sentenced to three years in prison, though some scholars argue he was a scapegoat for a broader research community that was quietly supportive.

Disability Critiques and "Velvet Eugenics"

• The Social Model View: Scholars Syfyan and Garland-Thompson argue that germline editing shifts the focus from "treating" people to "eliminating" types of people.

• Velvet Eugenics: A term used by Garland-Thompson to describe a "softer" form of eugenics enacted through market-driven reproductive choices and genetic testing rather than state-mandated sterilization.

• Core Critique: CRISPR based on a reductionist assumption that certain genetic differences are inherently negative and should be erased, which may devalue the identities and lives of people currently living with those conditions.

Clicker Questions:

The argument that CRISPR editing could cause harm if there are off-target effects fits best with which principle?

A. Justice

B. Beneficence

C. Non-maleficence (answer)

D. Respect for Autonomy

Sickle cell treatment process

This kind of gene therapy is classified as:

A. Ex vivo (answer)

B. In vivo

C. In situ

What, according to Garland-Thompson, is “velvet eugenics”?

A. A form of eugenics that endorses legal enforcement of restrictions on reproduction for some groups

B. Eugenics aimed at promoting reproduction among groups that are deemed to have desirable traits

C. A new form of eugenics that is promoted by market forces offering selection options as “choice” to consumers as opposed to government mandates (answer)