L2: Gene Therapy & Stem Cell Therapy

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What is gene therapy and nucleic acid therapeutics.

🔹Q: What is GENE THERAPY?

A treatment approach that uses genetic material to INTERFERE with cellular processes and treat disease w/ specificity.

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What is gene therapy and nucleic acid therapeutics.

🔹Q: What is NUCLEIC ACID THERAPEUTICS?

Therapies that use DNA or RNA molecules to INTERFERE w/ Gene expression or Cellular processes w/ high specificity.

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🔹Q: What is the central dogma of molecular biology?

Genetic info flows from:

• DNA → RNA → Proteins

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🔹Q: Why are Gene therapy and Nucleic acid therapeutics important?

They provide the capability to INTERFERE @ different stages of Cellular processes w/ ⬆HIGH specificity.

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Define replication, transcription, translational, and transformation.

🔹Q: What is Replication?

The process where double helical DNA unwinds & creates a COPY OF ITSELF so genetic information can be transferred to the next generation of cells or organism.

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🔹Q: What is Transcription?

The process where info stored in DNA is transferred to RNA.

• DNA → mRNA

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🔹Q: What is Translation?

The process where mRNA binds to ribosomes & the info in mRNA is read as triplet codons to assemble proteins.

• mRNA → Proteins

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🔹Q: What is Transformation?

The addition/incorporation of foreign DNA into a bacterium.

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🔹Q: What is the order of Genetic information flow?

DNA → RNA → Protein

⭐️Replication = copies DNA

⭐️Transcription = makes mRNA

⭐️Translation = makes Protein

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🔹Q: How is Transformation used in Biotech?

Scientists insert genes into plasmids, & bacteria take up the foreign DNA through Transformation to produce DESIRED proteins.

• Plasmids: small circular DNA molecule found in bacteria that replicated independently from chromosomal DNA.

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🧠Easy Memory Trick

• Replication

• Transcription

• Translation

• Transformation

• Replication = DNA → DNA

• Transcription = DNA → mRNA

• Translation = mRNA → Protein

• Transformation = foreign DNA enters BACTERIA

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Explain gene expression and its mechanism

• ANTIGENE mech

🔹Q: What is the ANTIGENE mech?

BLOCKS Gene Expression by preventing the unwinding of double helical DNA needed for transcription.

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Explain gene expression and its mechanism

• ANTIGENE mech

🔹Q: MOA of ANTIGENE?

A stable triple helix forms w/ DNA, which:

• PREVENTS DNA unwinding

• Or BLOCKS Transcription factor binding → blocking Transcription

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🔹Q: What does the ANTIGENE mechanism block?

Transcription of the targeted DNA region.

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🔹Q: What type of structure if formed in the ANTIGENE mechanism?

Triplex (a triple helix structure).

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🔹Q: What is the purpose of ANTIGENE therapy?

To selectively INHIBIT gene expression & PREVENT disease-related protein production.

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🔹Q: Simple explanation of ANTIGENE therapy?

ANTIGENE therapy “locks” DNA closed so it cannot be opened & copied into mRNA.

• ANTIGENE = against DNA Transcription (blocks DNA → mRNA)

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Explain gene expression and its mechanism

• ANTISENSE mech (2 types)

🔹Q: What is ANTISENSE mech?

Uses REVERSE-COMPLEMENTARY antisense oligonucleotides (ASOs) that bind (hybridize) to target mRNA & BLOCK gene expression.

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🔹Q: What are ANTISENSE Oligonucleotides (ASOs)?

ASOs = short synthetic nucleic acid strands designed to bind complementary target mRNA.

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🔹Q: What does ANTISENSE mean?

“Antisense” = Oligonucleotides sequence is COMPLEMENTARY to the target mRNA sequence.

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🔹Q: What is the 1st type of ANTISENSE mech?

ASOs bind to mRNA & sterically BLOCK expression, PREVENTING ribosomes from TRANSLATING the mRNA → Protein.

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🔹Q: What does steric blocking ANTISENSE therapy work?

ASOs physically block the ribosome from reading the mRNA.

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🔹Q: What is the 2nd type of ANTISENSE mech?

ASOs form an RNA-DNA duplex that becomes a substrate for RNase H enzyme → which CLEAVES the target mRNA.

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🔹Q: What does RNase H do in ANTISENSE therapy?

RNase H hydrolyzes (cleaves) the RNA strand of an RNA-DNA duplex → DESTROYING target mRNA.

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🔹Q: What is the overall goal of ANTISENSE mechs?

To INHIBIT Gene Expression by Blocking or Destroying target mRNA BEFORE Protein production occurs.

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🔹Q: Simple explanation of ANTISENSE therapy?

Antisense therapy sticks to mRNA sot eh cell either:

1. CANNOT read it, or

2. DESTROYS it

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🧠Easy memory: ANTISENSE

• Type 1

• Type 2

• Type 1 = block TRANSLATION

• Type 2 = RNase H CLEAVES mRNA

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Explain gene expression and its mechanism

• RNA INTERFERENCE mech

🔹Q: What is RNA INTERFERENCE (RNAi)?

A Gene-silencing mech that uses small interfering RNA (siRNA) to BLOCK gene expression by DESTROYING target mRNA.

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🔹Q: What is siRNA (small interfering RNA)?

Short SYNTHETIC double-stranded RNA molecule.

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🔹Q: What happens to siRNA AFTER entering the cell?

siRNA enters the cytoplasm & becomes incorporated into the RNA-Induced Silencing Complex (RISC).

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🔹Q: What does RISC do in RNA interference?

RISC = UNWINDS the double-stranded RNA & CLEAVES the target sense mRNA strand.

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🔹Q: What is the Overall result of RNA Interference?

Target mRNA is DESTROYED, preventing Protein productions & SILENCING Gene expression.

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🔹Q: How is RNA interference DIFFERENT from antisense therapy?

➡RNA Interference uses:

• siRNA

• RISC complex

• Cleavage of mRNA

➡Antisense therapy uses:

• ASOs to BLOCK or CLEAVE mRNA through RNase H.

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Describe two method of systemic delivery of nucleic acids.

• Viral delivery systems (Viral Vectors) – viral genetic material (Transduction)

🔹Q: What are VIRAL delivery systems (Viral vector)?

Viral delivery systems use viruses to deliver genetic material → into host cell.

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• Viral delivery systems (Viral Vectors) – viral genetic material (Transduction)

🔹Q: What is Transduction?

The process where VIRAL VECTORS deliver Genetic Material → into cells.

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🔹Q: Why are VIRAL VECTORS used in Gene Therapy?

Viruses naturally enters cells efficiently → making them useful for delivering THERAPEUTIC Genes

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🔹Q: What are examples of VIRAL VECTORS?

• Retroviruses

• Adenoviruses

• Adenovirus-associated viruses (AAV)

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🔹Q: What type of Genetic Material do Retroviruses contain?

SINGLE-stranded RNA genetic material.

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🔹Q: What type of Genetic Material do Adenoviruses contain?

DOUBLE-stranded DNA genetic material.

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🔹Q: What type of Genetic Material do AAV (adeno-associated viruses) contain?

SINGLE-stranded DNA genetic material.

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🔹Q: Simple explanation of VIRAL VECTORS?

Modified viruses used as “Delivery Trucks” to carry & transport THERAPEUTIC genes → into cells.

🧠Virus = Vector = Transduction

🧠Viral delivery = gene enters cells efficiently using a virus

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Describe two method of systemic delivery of nucleic acids.

• Non-viral delivery systems (Synthetic Vectors) – lipoplex, polyplex (Transfection)

🔹Q: What are NON-VIRAL Delivery systems (Synthetic vectors)?

Non-viral Delivery systems use SYNTHETIC material to deliver DNA or RNA → into cells INSTEAD of viruses.

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• Non-viral delivery systems (Synthetic Vectors) – lipoplex, polyplex (Transfection)

🔹Q: What is Transfection?

Process of delivering Nucleic acids → into cells using NON-VIRAL methods.

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🔹Q: How do SYNTHETIC Vectors bind Nucleic acids?

Synthetic vectors electrostatically bind (-)negatively charged DNA or RNA & condense them → into nanosized particles.

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🔹Q: Why is DNA negatively charged?

B/c of PHOSPHATE groups in Nucleotides.

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🔹Q: What is a Lipoplex?

A Lipoplex forms when CATIONIC lipids (or Cationic liposomes) complex w/ DNA.

• DNA = (-)

• Cationic lipids = (+)

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🔹Q: What is used to form Lipoplexes?

(CATIONIC Lipids / CATIONIC Liposomes) + (DNA)

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🔹Q: What is a Polyplex?

A Polyplex forms when CATIONIC polymers (or polypeptides) complex w/ DNA.

• DNA = (-)

• CATIONIC polymers = (+)

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🔹Q: What is used to form a Polyplexes?

(CATIONIC polymers / polypeptides) + (DNA)

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🔹Q: What is the Purpose of NON-VIRAL delivery systems?

To TRANSPORT Therapeutic nucleic acids → Into cells w/out using viruses.

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🔹Q: Simple explanation of TRANSFECTION?

Uses SYNTHETIC carriers to “Package & Deliver” DNA/RNA → into Cells.

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🧠Lipoplex vs. Polyplex?

➡Lipoplex = LIPID + DNA

➡Polyplex = POLYMER + DNA

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🧠NON-VIRAL delivery?

Synthetic vector = Transfection

⭐️NON-viral delivery uses ARTIFICIAL carriers instead of viruses.

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Explain gene-based therapy.

• Gene REPLACEMENT therapy

🔹Q: What is GENE REPLACEMENT therapy?

Provides cells w/ a NEW, working copy of a missing or non-working gene.

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🔹Q: What is the goal of GENE REPLACEMENT therapy?

To 🔨RESTORE production of a NORMAL functional Protein by REPLACING a defective gene.

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🔹Q: How is GENE REPLACEMENT Therapy performed?

1. A working gene is created in Lab.

2. The gene is placed into a Vector (“Delivery Truck”)

3. The Vector delivers the gene → into the Nucleus of Target cells

4. The NEW gene begins producing the needed protein.

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🔹Q: What happens to the Therapeutic Gene AFTER delivery?

The gene may:

• Become part of the cell’s DNA, or

• Remain Separate, BUT it still produces the NEEDED protein.

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🔹Q: What happens to the Vector AFTER therapy?

When Vector is no longer needed = it is ELIMINATED from the body.

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🔹Q: Simple Explanation of GENE REPLACEMENT Therapy?

Gives a HEALTHY copy of a Broken gene so the cell can make the CORRECT PROTEIN again.

🧠Broken gene out → WORKING gene in

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Explain gene-based therapy.

• Gene EDITING

🔹Q: What is GENE EDITING?

A technique that INSERTS, REMOVES, or REPLACES specific pieces of a person’s DNA.

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🔹Q: What is the goal of GENE EDITING?

To change existing genes & CORRECT mutations @ PRECISE locations in DNA.

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🔹Q: What is the most important example of gene editing discussed in lecture?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats).

⭐️CRISPR = used to CHANGE DNA sequences & GENE function.

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🔹Q: Simple explanation of GENE EDITING?

Acts like “Molecular Scissors/Surgery” that cut & repair defective DNA.

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Explain gene-based therapy.

• CAR T-cell therapy

🔹Q: What is CAR T-cell therapy?

A type of CANCER IMMUNOTHERAPY that uses patient’s OWN T-cells to treat CANCER.

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🔹Q: What does CAR stand for in CAR T-cell therapy?

CHIMERIC Antigen Receptor.

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🔹Q: How does CAR T-cell therapy work?

1. T-cells are REMOVED from the patient’s blood.

2. A CAR gene is added to the T-cells.

3. The modified T-cells are MULTIPLIED.

4. CAR T-cells INFUSED BACK into the patient.

5. CAR receptors recognize & attack CANCER cells.

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🔹Q: What is the Purpose of CAR receptor?

CAR receptor binds proteins on Cancer cells & REDIRECT T-cells to target tumor-associated Antigens.

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🔹Q: What Cancers were mentioned for CAR T-cell therapy?

• B-cell Acute Lymphoblastic Leukemia (ALL)

• Large B-cell lymphoma

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🔹Q: What was the 1st FDA-approved CAR T-cell therapy?

Kymriah = for pediatric & young adult patients w/ ALL (Acute Lymphoblastic Leukemia).

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🔹Q: What is Yescarta?

A CAR T-cell therapy approved for certain adult patients w/ Large B-cell Lymphoma.

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🔹Q: What is the main idea behind CAR T-cell therapy?

It activates & REDIRECTS the body’s OWN T-cells to kill Cancer cells.