BPS3021 Chap.8 Nucleic Acid Therapeutics

Nucleic Acid Therapeutics

Match disease mechanism to expression, silencing or editing, then trace cargo to its required compartment.

What this chapter covers

• 01

  DNA and mRNA expression

• 02

  RNA Interference and siRNA Knockdown

• 03

  CRISPR-Based Correction

• 04

  Viral and Non-Viral Delivery

Worked example · free

Design a sequence-specific knockdown

Key terms

Gene replacement

Delivery of nucleic acid intended to restore expression of a missing or insufficient functional product.

RNA interference

Sequence-guided reduction of target RNA through cellular silencing machinery.

Genome editing

Directed alteration of genomic DNA using programmable editing machinery.

Nucleic Acid Therapeutics FAQ

Exam move

Answer in the order objective, cargo, destination, duration, limitations, measurements and controls; Nucleic acids can act as drugs by inducing expression, silencing a target or correcting a sequence; The biological problem determines the action;

Loss of a functional protein may motivate DNA or mRNA replacement; excess harmful protein may motivate siRNA knockdown; a defined pathogenic variant may motivate editing when the delivery and safety case is credible; DNA → nucleusmRNA → cytoplasmsiRNA → RISCCRISPR → genomeCargo choice determines the intracellular destination and the barriers that matter.Destination controls delivery difficultyDNA is large and hydrophilic, must escape endosomes and reach the nucleus, and requires regulatory elements for expression;

mRNA avoids the nuclear barrier but remains vulnerable to degradation and must reach the cytoplasm; siRNA also needs cytosolic access to guide sequence-specific silencing; CRISPR systems must deliver editing machinery to the appropriate compartment and then manage off-target and durability risks;

mRNA expression and siRNA knockdown are generally transient, so repeat dosing may be needed; Non-integrating DNA expression can also fade; A successful genomic edit may persist in the edited cell, but clinical benefit depends on which cells were edited, how many, whether they survive and whether function is restored;

Do not call an edit a permanent cure without those conditions; Viral vectors exploit evolved entry pathways but bring cargo, immunity, tropism and manufacturing constraints; Non-viral systems such as lipid nanoparticles can be engineered and redosed in some settings but still face extracellular loss, cell targeting, endosomal escape and toxicity;

The correct comparison is indication-specific, not a universal ranking; An original disease scenario involves excessive expression of a harmful protein with no need to alter DNA; A sequence-specific siRNA strategy is conceptually direct: deliver siRNA to the cytoplasm, load the guide into the silencing machinery, reduce target mRNA and measure both transcript and protein;

The response is transient, so dosing and duration must be evaluated; A missing secreted protein should be restored without editing the genome; Answer: mRNA replacement can provide transient expression after cytosolic delivery;

Measure encoded protein and function, while testing innate immune activation and durability; Labelled cargo shows high cell-associated fluorescence; Answer: this supports association only; Add extracellular quenching, localisation, cytosolic-access and functional readouts before claiming delivery;

The workshop allocates marks to strategy choice, molecular mechanism, duration, limitations and interpretation; Preserve that structure; Weak answers advertise a platform; strong answers establish a causal chain and state one conclusion the data cannot support;

End with the experiment that best separates competing explanations, such as uptake without escape versus escape without intact cargo; State why reducing expression addresses the disease mechanism; Name siRNA as the cargo and cytoplasm as the required destination;

Use a non-targeting sequence, untreated control and viability measurement; Measure target mRNA, target protein and a relevant functional response; Discuss off-target effects, delivery selectivity and repeat dosing; · CARGO + DESTINATION Therapeutic strategy WEEK 11 Match action to cargo and destination Expression, silencing and editing solve different biological problems Nucleic acids can act as drugs by inducing expression, silencing a target or correcting a sequence;

The biological problem determines the action; Loss of a functional protein may motivate DNA or mRNA replacement; excess harmful protein may motivate siRNA knockdown; a defined pathogenic variant may motivate editing when the delivery and safety case is credible;

therapeutic objective DNA → nucleus mRNA → cytoplasm siRNA → RISC CRISPR → genome Cargo choice determines the intracellular destination and the barriers that matter; Destination controls delivery difficulty DNA is large and hydrophilic, must escape endosomes and reach the nucleus, and requires regulatory elements for expression;

mRNA avoids the nuclear barrier but remains vulnerable to degradation and must reach the cytoplasm; siRNA also needs cytosolic access to guide sequence-specific silencing; CRISPR systems must deliver editing machinery to the appropriate compartment and then manage off-target and durability risks;

Duration is not the same as benefit mRNA expression and siRNA knockdown are generally transient, so repeat dosing may be needed; Non-integrating DNA expression can also fade; A successful genomic edit may persist in the edited cell, but clinical benefit depends on which cells were edited, how many, whether they survive and whether function is restored;

Do not call an edit a permanent cure without those conditions; ANSWER SEQUENCE Disease mechanism → therapeutic action → cargo → destination → duration → limitations and measurements; Viral vectors exploit evolved entry pathways but bring cargo, immunity, tropism and manufacturing constraints;

Non-viral systems such as lipid nanoparticles can be engineered and redosed in some settings but still face extracellular loss, cell targeting, endosomal escape and toxicity; The correct comparison is indication-specific, not a universal ranking;

Cell selectivity determines whether delivery is therapeutic or merely efficient; Expression in an unintended tissue can create toxicity even when cargo reaches the correct intracellular compartment; Include biodistribution and cell-type specificity beside molecular delivery in the development plan;

· · JUSTIFY THE STRATEGY Strategy justification Separate uptake from productive action Mechanism, duration and safety must all appear in the recommendation EX 8 Choose a knockdown strategy An original disease scenario involves excessive expression of a harmful protein with no need to alter DNA;

A sequence-specific siRNA strategy is conceptually direct: deliver siRNA to the cytoplasm, load the guide into the silencing machinery, reduce target mRNA and measure both transcript and protein; The response is transient, so dosing and duration must be evaluated;

State why reducing expression addresses the disease mechanism; Name siRNA as the cargo and cytoplasm as the required destination; Use a non-targeting sequence, untreated control and viability measurement; Measure target mRNA, target protein and a relevant functional response;

Discuss off-target effects, delivery selectivity and repeat dosing; P1 mRNA replacement Practice A missing secreted protein should be restored without editing the genome; Answer: mRNA replacement can provide transient expression after cytosolic delivery;

Measure encoded protein and function, while testing innate immune activation and durability; P2 Fluorescence boundary Practice Labelled cargo shows high cell-associated fluorescence; Answer: this supports association only;

Add extracellular quenching, localisation, cytosolic-access and functional readouts before claiming delivery; A credible gene-therapy answer names where the cargo must go and what result would prove that it worked; Nucleic-acid closing move The workshop allocates marks to strategy choice, molecular mechanism, duration, limitations and interpretation;

Preserve that structure; Weak answers advertise a platform; strong answers establish a causal chain and state one conclusion the data cannot support; End with the experiment that best separates competing explanations, such as uptake without escape versus escape without intact cargo;

Durability should be measured rather than inferred from platform labels; Follow the molecular change, functional response and relevant cell population over time; A declining cargo signal with sustained function may mean something different from persistent cargo without a biological response; ·