Biologics by product type: antisense therapy MCQs With Answer

Introduction: Biologics by product type: antisense therapy MCQs With Answer is a focused quiz set designed for M.Pharm students studying Biological Evaluation of Drug Therapy. This collection covers fundamental and advanced concepts of antisense oligonucleotide (ASO) therapeutics — including mechanism of action, chemical modifications, delivery strategies, pharmacokinetics, safety issues and analytical evaluation. Questions emphasize clinically relevant examples, design principles, bioanalytical methods and regulatory considerations to prepare students for examinations and research applications. Each MCQ tests applied understanding rather than memorization, helping students integrate molecular-level knowledge with pharmacological and translational aspects of antisense biologics.

Q1. What is the primary mechanism by which a gapmer antisense oligonucleotide reduces target mRNA levels?

• Steric blocking of ribosome assembly on mRNA
• Recruitment of RNase H to cleave the RNA strand of RNA–DNA duplex
• Interference with pre-mRNA splicing by blocking splice sites
• Activation of RNA interference (RISC) complex to degrade mRNA

Correct Answer: Recruitment of RNase H to cleave the RNA strand of RNA–DNA duplex

Q2. Which chemical backbone modification is most commonly associated with increased plasma protein binding and nuclease resistance in first‑generation ASOs?

• 2′-O-methyl ribose
• Phosphorothioate (PS) backbone
• Peptide nucleic acid (PNA)
• Phosphodiester backbone

Correct Answer: Phosphorothioate (PS) backbone

Q3. Which antisense drug modality is primarily designed to alter pre-mRNA splicing and is used for exon skipping or inclusion therapies?

• Gapmer ASO recruiting RNase H
• Steric‑blocking splice switching oligonucleotide (SSO)
• siRNA delivered via RISC
• Antibody–oligonucleotide conjugate

Correct Answer: Steric‑blocking splice switching oligonucleotide (SSO)

Q4. Which of the following oligonucleotide chemistries is charge-neutral and commonly used for splice‑modulating steric block ASOs with high nuclease stability?

• 2′-O-methoxyethyl (2′-MOE)
• Phosphorothioate (PS)
• Peptide nucleic acid (PNA)
• Phosphodiester RNA

Correct Answer: Peptide nucleic acid (PNA)

Q5. Nusinersen, an FDA‑approved antisense therapeutic for spinal muscular atrophy (SMA), is administered by which route to achieve therapeutic CNS concentrations?

• Oral
• Intravenous (IV)
• Intrathecal
• Subcutaneous (SC)

Correct Answer: Intrathecal

Q6. Conjugation of antisense oligonucleotides to N‑acetylgalactosamine (GalNAc) primarily improves delivery to which organ?

• Liver (hepatocytes)
• Spleen macrophages
• Central nervous system neurons
• Lungs (alveolar epithelium)

Correct Answer: Liver (hepatocytes)

Q7. Which adverse effect is particularly associated with some phosphorothioate-modified ASOs and can be monitored clinically?

• QT interval prolongation only
• Thrombocytopenia
• Severe hypoglycemia
• Pulmonary fibrosis exclusively

Correct Answer: Thrombocytopenia

Q8. Which bioanalytical technique is most appropriate for sequence‑specific quantitation of intact antisense oligonucleotides in plasma?

• Western blot
• Hybridization ELISA or Hybridization LC-MS/MS
• Colorimetric Bradford assay
• Reverse transcriptase PCR (RT-PCR) for protein

Correct Answer: Hybridization ELISA or Hybridization LC-MS/MS

Q9. Which molecular feature most directly increases an ASO’s binding affinity (higher Tm) for its complementary RNA target?

• Shorter oligonucleotide length
• Inclusion of locked nucleic acids (LNA) or constrained 2′ modifications
• Use of unmodified ribose with phosphodiester linkage
• Reduction of GC content

Correct Answer: Inclusion of locked nucleic acids (LNA) or constrained 2′ modifications

Q10. A steric‑blocking antisense oligonucleotide that lacks RNase H activity is expected to produce which result?

• Direct cleavage of target mRNA by RNase H
• Block access of splicing factors or ribosomes without mRNA cleavage
• Recruitment of Dicer and RISC complex
• Permanent genomic DNA editing

Correct Answer: Block access of splicing factors or ribosomes without mRNA cleavage

Q11. Which of the following best explains why phosphorothioate ASOs often show broad tissue distribution but also higher non‑specific protein interactions?

• PS backbone creates a neutral molecule that diffuses freely
• PS substitutions increase hydrophobicity and negative charge interactions with serum proteins
• PS backbone is rapidly degraded, forcing accumulation in tissues
• PS oligos are converted into lipophilic metabolites in vivo

Correct Answer: PS substitutions increase hydrophobicity and negative charge interactions with serum proteins

Q12. For antisense ASOs that act via RNase H, what is the typical design strategy for the central region of the oligonucleotide?

• Use of bulky, fully modified nucleotides in the center to prevent cleavage
• Incorporation of a DNA gap (deoxy residues) flanked by modified nucleotides
• All ribonucleotides to maximize RNase resistance
• Conjugation to peptides in the central region to enhance uptake

Correct Answer: Incorporation of a DNA gap (deoxy residues) flanked by modified nucleotides

Q13. Which immunostimulatory risk should be considered during antisense sequence selection, especially if unmethylated CpG motifs are present?

• Activation of Toll‑like receptor 9 (TLR9) leading to innate immune responses
• Specific activation of TLR3 only
• Exclusive B‑cell mediated antibody production against target mRNA
• Complete absence of any immune activation due to sequence

Correct Answer: Activation of Toll‑like receptor 9 (TLR9) leading to innate immune responses

Q14. Which of the following clinical antisense drugs is an example of exon skipping therapy for Duchenne muscular dystrophy?

• Fomivirsen
• Eteplirsen
• Nusinersen
• Inotersen

Correct Answer: Eteplirsen

Q15. During preclinical pharmacokinetic studies, what is the predominant elimination route for small ASOs (<20 kDa) with PS backbones?

• Hepatobiliary excretion unchanged primarily
• Renal filtration and urinary excretion of intact and metabolized oligos
• Exhalation via lungs after metabolic conversion
• Complete intracellular degradation with no systemic excretion

Correct Answer: Renal filtration and urinary excretion of intact and metabolized oligos

Q16. Which analytical impurity class in antisense oligonucleotide manufacture refers to shortened products missing one or more nucleotides?

• Deamidation products
• Shortmers (n-1, n-2 species)
• Cross‑linked dimers
• Sulfated adducts

Correct Answer: Shortmers (n-1, n-2 species)

Q17. What is “gymnosis” in the context of antisense oligonucleotide studies?

• Endosomal escape facilitated by cell-penetrating peptides
• Uptake of naked ASOs by cells without transfection reagents in vitro
• Conjugation to GalNAc for hepatic delivery
• Cleavage of ASO by RNase H in the nucleus

Correct Answer: Uptake of naked ASOs by cells without transfection reagents in vitro

Q18. Which modification is commonly used at the 2′ position of ribose to improve nuclease resistance and reduce toxicity while maintaining binding affinity?

• 2′-O-methoxyethyl (2′-MOE)
• 5-methylcytosine only
• Unmodified 2′-hydroxyl
• Phosphodiester linkage at each internucleotide position

Correct Answer: 2′-O-methoxyethyl (2′-MOE)

Q19. In designing antisense sequences, which parameter most directly reduces hybridization off‑target risk?

• Choosing sequences with many repetitive motifs
• Maximizing sequence uniqueness across the transcriptome and avoiding partial complementarity
• Selecting sequences with highest GC content regardless of uniqueness
• Using the shortest possible oligo (5–6 mer) to reduce length

Correct Answer: Maximizing sequence uniqueness across the transcriptome and avoiding partial complementarity

Q20. Which preclinical assay is most useful to demonstrate that an ASO’s activity is RNase H‑dependent rather than steric blocking?

• Measure ASO binding to albumin in plasma
• Compare target mRNA reduction in cells using RNase H knockout or inhibition versus wild type
• Assess antibody titers against the ASO sequence
• Evaluate melting temperature (Tm) only

Correct Answer: Compare target mRNA reduction in cells using RNase H knockout or inhibition versus wild type

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