Q1.

Critically evaluate the drug delivery requirements for BOTH successful RNA
interference treatment and RNA mediated vaccination. Please use named examples.
Identify factors that prevent siRNA medicines from becoming mainstream (25% of the
available marks)

RNA Interference (RNAi) Treatments

Mechanism and Application

RNA interference (RNAi) therapy entails producing tiny interfering RNAs (siRNAs) that
match a gene's mRNA sequence. These siRNAs are delivered into cells and integrated into
the RNA-induced silencing complex (RISC). The RISC-siRNA complex attaches to the target
mRNA, causing cleavage and destruction. This technique effectively silences the gene,
inhibiting the creation of the corresponding protein, which may reduce illness symptoms or
progression by targeting genes associated with certain disorders. This gene silencing
mechanism has shown promise in addressing diseases by knocking down genes associated
with pathology.

Named Example

Patisiran (Onpattro®) is an FDA-approved siRNA treatment for hereditary transthyretin-

mediated amyloidosis that delivers siRNA to liver cells via lipid nanoparticles. It silences the
defective transthyretin gene, demonstrating siRNA's promise for treating genetic disorders
(Bumcrot et al., 2006).

Factors Preventing siRNA Mainstream Use

 Stability: RNA's susceptibility to degradation by nucleases.

 Delivery: The necessity for effective delivery mechanisms.

 Specificity: The risk of off-target effects and unintended gene silencing.

 Immunogenicity: Potential activation of immune responses.

 Cost: High development and manufacturing expenses (Bumcrot et al., 2006).

RNA-mediated Vaccination

Mechanism and Application

RNA-mediated vaccination entails injecting messenger RNA (mRNA), which encodes for a
pathogen's antigen. Once within the body, cells pick up the mRNA and convert it into the
antigenic protein. The immune system perceives these proteins as alien and initiates an
immunological response, which includes the production of antibodies. This process prepares
the immune system to recognize and combat the virus in future encounters. Notably, mRNA
vaccines, such as those produced for COVID-19, employ lipid nanoparticles to preserve the
mRNA and allow it to enter cells.

Named Example

The Pfizer-BioNTech and Moderna COVID-19 vaccines are prominent examples of mRNA
vaccines encoding the SARS-CoV-2 spike protein. These vaccines utilize lipid nanoparticles
for mRNA protection and cell entry facilitation, illustrating the practical application of RNA
technology in vaccination.

Factors Preventing RNA Vaccines Mainstream Use

 Cold Chain: High storage and transportation temperatures requirements.

 Public Perception: Challenges in overcoming vaccine hesitancy due to

misinformation.

 Production Challenges: Specialized and complex manufacturing processes.

 Duration of Protection: Ongoing research into the long-term efficacy of mRNA

vaccines.

 Novelty: Establishing long-term safety data and building public trust in this new
technology.

Yang et al. (2020) demonstrated siRNA's potential against Trichinella spiralis, suggesting
wider disease applicability. Bumcrot et al. (2006) emphasized RNA technology's public
health impact through COVID-19 vaccines, highlighting the challenges and opportunities in
public acceptance of RNA-based therapies.

Q2. Casgevity (exagamglogene autotemcel) represents a first in class drug. Critically

evaluate its mode of action and describe some of the delivery barriers that this medicine
has had to overcome to be clinically effective. Critically evaluate why this approach is
limiting. (25% of the available marks)
Exagamglogene autotemcel (exa-cel), uses CRISPR/Cas9 technology to treat transfusion-
dependent β-thalassemia (TDT) by modifying autologous CD34+ HSPCs. To compensate for
adult hemoglobin deficiency, it targets the BCL11A gene's erythroid-specific enhancer region
ex vivo to revive fetal hemoglobin (HbF) synthesis. This one-time, non-viral cell treatment
may cure TDT by maintaining high HbF levels. Clinical investigations show that most
patients achieve transfusion independence and early and persistent Hb and HbF rises.
However, gene editing and autologous stem cell transplantation need specialist facilities,
making the treatment difficult to get. Interim findings are promising, but exact genetic
changes' long-term effectiveness and safety need more study (Locatelli et al., 2023; Mapara,
2024).

In autologous CD34+ hematopoietic stem and progenitor cells, Exa-cel targets the BCL11A
gene's erythroid-specific enhancer region. Adults need BCL11A to decrease fetal hemoglobin
(HbF) synthesis. By altering this enhancer area, exa-cel downregulates BCL11A, reactivating
red blood cell HbF synthesis.

The ex vivo manipulation approach comprises modifying patient's HSPCs using

CRISPR/Cas9 to stimulate HbF production, then reinfusing the changed cells back into the
patient. This method requires myeloablative conditioning to provide room for altered cells in
the patient's bone marrow.

The main treatment objective is to boost HbF levels to compensate for the deficient adult
hemoglobin in β-thalassemia. HbF has a greater oxygen affinity than adult hemoglobin,
making it beneficial even in severe β-thalassemia patients, possibly lowering or eliminating
the requirement for blood transfusions.

Exagamglogene autotemcel (exa-cel) has major delivery issues in treating transfusion-

dependent β-thalassemia (TDT). A critical challenge was assuring CRISPR/Cas9's efficacy in
editing the BCL11A gene in autologous CD34+ HSPCs, which required improved lab
procedures to achieve high gene-edit success rates (Locatelli et al., 2023; Mapara et al.,
2024). After editing, the HSPCs needed myeloablative conditioning to successfully engraft in
the patient's bone marrow, overcoming natural hurdles to cell implantation. The therapy's
efficacy is dependent on the survival and multiplication of these transformed cells.
Furthermore, accurate CRISPR/Cas9 component design was required to reduce off-target
effects while ensuring the safety of viral vectors employed in delivery. Ex vivo modification
of HSPCs is difficult and labor-intensive, necessitating uniform protocols across treatment
facilities to ensure cell survival and purity (Locatelli et al., 2023; Mapara et al., 2024).
Overcoming these hurdles was critical to exa-cel's clinical success in obtaining transfusion
independence in TDT patients.

Q3. RAS is one of the most commonly mutated oncogenes in cancer. Critically evaluate
the statement: "RAS is undruggable".

Cancer therapy has long struggled to target RAS, a key oncogene often altered in many
malignancies. Due to their strong affinity for GTP and absence of small molecule binding
sites, RAS proteins have been considered "undruggable." The technical challenges of
inhibiting the RAS-GTP connection, which is necessary for its carcinogenic activity, led to
this assumption (Cox et al., 2014). Recent scientific breakthroughs have challenged this idea,
revealing novel direct and indirect RAS cancer treatment options.

Understanding RAS's molecular structure and finding allosteric sites has revived direct
targeting. Researchers found tiny compounds that may bind to RAS at these unique locations,
blocking its interaction with downstream effectors like RAF. These results suggest that RAS
may have undiscovered pharmacological vulnerabilities that might be used therapeutically.

In addition, mutant-specific inhibitors are potential for RAS-targeted treatment. Compounds

that specifically block RAS mutations like KRAS G12C have shown that precision medicine
can cure RAS-driven malignancies.

Instead of directly inhibiting RAS, addressing downstream signaling pathways including the
RAF-MEK-ERK and PI3K-AKT pathways is an alternate technique. Researchers hope to
disrupt these pathways to limit mutant RAS's oncogenic signals, preventing cancer cell
growth and survival. Several pathway inhibitors have been clinically developed using this
method, demonstrating the potential for indirect RAS-targeted therapies.

These improvements change the view of RAS as a "undruggable" target. RAS-targeted cancer
therapeutics may now be developed using direct inhibitors, mutation-specific targeting, and
downstream effector pathway inhibition. RAS biology is evolving, and new treatments that
question the long-held concept of its undruggability are becoming possible, bringing RAS-
driven cancers closer to viable treatments (Cox et al., 2014).

Q4 Critically evaluate CAR T cell therapy in both blood cancers and in solid tumours.
Include named examples.
CAR T-cell therapy, a groundbreaking method in cancer treatment, has shown impressive
results in blood cancers but encounters significant obstacles in solid tumors. Hematological
cancers have seen significant advancements in treatment with the introduction of CAR T-cell
therapies such as Kymriah (Tisagenlecleucel) for B-ALL and Yescarta (Axicabtagene
ciloleucel) for DLBCL. These therapies have demonstrated remarkable effectiveness,
resulting in their approval by the FDA. These therapies utilize genetically modified T cells to
specifically target antigens found in cancer cells, leading to high rates of remission in cases
that were previously resistant or had relapsed (Ma et al., 2019).

The progress of CAR T-cell therapy's effectiveness in treating solid tumors has been impeded
by various biological and technical challenges. The intricate microenvironment of solid
tumors presents formidable challenges for CAR T-cell infiltration, persistence, and activity.
This environment is marked by immunosuppressive cells and cytokines, physical barriers
such as the extracellular matrix, and the diverse array of tumor antigens (Ma et al., 2019;
Dana et al., 2021). In addition, the challenge of developing effective CAR T-cell therapies for
solid tumors is compounded by the potential for on-target/off-tumor toxicity caused by the
shared expression of target antigens between tumor and normal tissues.

Efforts to address these challenges involve the modification of CAR T cells to express
chemokine receptors that align with those produced by tumors, thereby improving their
ability to travel and infiltrate (Ma et al., 2019). In addition, researchers are currently
exploring strategies that involve dual CAR systems to target multiple antigens and include
modules to counteract the immunosuppressive tumor microenvironment. However, despite
these advancements and the initiation of numerous clinical trials, particularly in China, there
has yet to be a major development in the clinical approval of CAR T-cell therapies for solid
tumors (Ma et al., 2019).

Overall, CAR T-cell therapy has shown great promise in the treatment of hematological
malignancies, but its effectiveness in solid tumors is still being studied and improved.
Addressing the distinct obstacles posed by the tumor microenvironment, prioritizing precise
targeting to reduce potential harm, and improving the endurance and effectiveness of CAR T-
cell therapy are crucial focal points of current scientific investigation. For solid tumors,
achieving successful CAR T-cell therapies will likely necessitate a blend of inventive
engineering approaches and a more profound comprehension of tumor biology (Ma et al.,
2019; Dana et al., 2021).
References

Cox, A.D., Fesik, S.W., Kimmelman, A.C., Luo, J. and Der, C.J., 2014. Drugging the
undruggable RAS: Mission possible?. Nature reviews Drug discovery, 13(11), pp.828-851.

Ma, S., Li, X., Wang, X., Cheng, L., Li, Z., Zhang, C., Ye, Z. and Qian, Q., 2019. Current
progress in CAR-T cell therapy for solid tumors. International journal of biological
sciences, 15(12), p.2548.

Dana, H., Chalbatani, G.M., Jalali, S.A., Mirzaei, H.R., Grupp, S.A., Suarez, E.R., Rapôso,
C. and Webster, T.J., 2021. CAR-T cells: Early successes in blood cancer and challenges in
solid tumors. Acta Pharmaceutica Sinica B, 11(5), pp.1129-1147.

Yang, F., Guo, K.X., Yang, D.Q., Liu, R.D., Long, S.R., Zhang, X., Jiang, P., Cui, J. and
Wang, Z.Q., 2020. Functional analysis of Trichinella spiralis serine protease 1.2 by siRNA
mediated RNA interference.

Bumcrot, D., Manoharan, M., Koteliansky, V. et al., 2006. RNAi therapeutics: a potential
new class of pharmaceutical drugs. Nat Chem Biol, 2, pp.711–719.
https://doi.org/10.1038/nchembio839
Locatelli, F., Lang, P., Corbacioglu, S., Wall, D., Meisel, R., Li, A.M., de La Fuente, J.,
Shah, A.J., Carpenter, B., Kwiatkowski, J.L. and Mapara, M., 2023. Exagamglogene
Autotemcel for Transfusion-Dependent β-Thalassemia. Blood, 142, p.1053.

Mapara, M.Y., Locatelli, F., Lang, P., Corbacioglu, S., Li, A., de la Fuente, J., Wall, D.A.,
Meisel, R., Shah, A.J., Liem, R. and Carpenter, B., 2024. Transfusion Independence after
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Thalassemia. Transplantation and Cellular Therapy, 30(2), p.S236.