Repair and Regeneration

What are the 3 types of non-cell based therapeutics?

Acellular scaffolds, pharmacological small molecules, in vivo gene editing.

What is the advantage of gene editing over traditional gene therapy?

Gene editing can permanently alter the genome, avoiding issues like limited cargo size, transient effects, and oncogenic risks of traditional gene therapy.

What is CRISPR/Cas9 and how does it work?

CRISPR/Cas9 introduces double-strand breaks at specific genomic sites using guide RNAs (near PAM), allowing the cell’s repair machinery to introduce mutations or corrections (NHEJ deletes and HDR inserts)

What is transthyretin amyloidosis (TTR amyloidosis)?

A life-threatening disease caused by the accumulation of misfolded TTR protein, mainly affecting the nerves and heart.

What is the goal of in vivo gene editing for TTR amyloidosis?

To delete the TTR gene in liver cells, preventing production of misfolded transthyretin and halting disease progression.

How is CRISPR-Cas9 delivered for in vivo editing in TTR amyloidosis patients?

Using lipid nanoparticles (LNPs) decorated with ApoE, which targets LDL receptors on hepatocytes.

What happens to the TTR gene after CRISPR-Cas9 editing?

The gene is scrambled by NHEJ, making the mRNA non-functional and untranslatable.

What were the clinical results of TTR in vivo CRISPR-Cas9 therapy?

Up to 90% reduction in serum TTR levels within 28 days of a single dose

What is dCas9 and how does it differ from regular Cas9?

dCas9 (dead Cas9) has no endonuclease activity and is used to regulate gene expression rather than DNA cleavage.

How can dCas9 be used to control gene expression?

It can be fused to a repressor or activator domain and directed to specific gene loci using guide RNAs to silence or activate transcription.

What are the 2 dCas9 strategies?

Direct fusion or modular strategy.

Explain direct fusion dCas9.

A repressor or activator domain is directly fused to dCas9 as a single polypeptide chain (one construct per dCas9).

What is the repressor dCas9 fusion?

DCas9-KRAB

What is the activator dCas9 fusion?

DCas9-VP64.

Explain modular dCas9.

dCas9 is tagged with a recruitment handle/hairpins (like MS2, SunTag) so effectors can be switched (KRAB or VP64).

What is base editing?

Base editing is a gene-editing technique that enables the precise, irreversible conversion of one DNA base to another without cutting either strands of DNA.

What are the two main components of a base editor?

A modified Cas9 (dCas9/Cas9 nickase) fused to a deaminase enzyme.

What is a cytosine base editor (CBE)?

A base editor that converts cytosine (C) to thymine (T), turning a CG base pair into a TA pair (e.g., APOBEC1).

What is an adenine base editor (ABE)?

A base editor that converts adenine (A) to guanine (G), changing an AT base pair into a GC pair (e.g., ABE7.10).

What is Cas9 nickase?

It is a modified version of Cas9 where one of the two nuclease domains (HNH or RuvC) has been mutated to render it non-functional, effectively making it a nickase. This allows it to create a single-strand break (a "nick") in the target DNA, rather than a double-strand break (DSB) like the standard Cas9. 

What is the “editing window” in base editing?

The specific region within the DNA sequence where the base conversion can occur, guided by the sgRNA.

What diseases could base editing potentially treat?

Disease causing point-mutations, such as sickle cell anemia.

How does prime editing work?

It uses a Cas9 nickase fused to a reverse transcriptase and guided by a prime editing guide RNA (pegRNA). The pegRNA directs the complex to the target DNA site, where the Cas9 nickase creates a single-strand nick. The reverse transcriptase then copies the desired edit from the pegRNA into the DNA. Cellular repair mechanisms incorporate the edit into both DNA strands, achieving precise modifications without double-strand breaks.

Why are miRNAs relevant in cancer therapy?

miRNAs regulate gene expression and are often downregulated in cancers, where they normally control cell proliferation, apoptosis, and immune responses.

What is miRNA-34a, and why is it significant?

miR-34a is downregulated in >50% of cancers and normally suppresses cancer cell proliferation, migration, and immune evasion making it a therapeutic target.

What 2 types of miRNA therapies can enhance expression?

Mimics and viral vector delivery.

What 4 types of miRNA therapies inhibit miRNA expression?

Anti-miRNAs, sponges, masks, decoys.

What is a major challenge with miRNA-based therapies?

miRNAs are unstable in serum and are rapidly degraded, so they can’t be directly injected.

How can miR-34a stability and targeting be improved?

Chemical modification which attaches a folate tag to target overexpressed folate receptors on cancer cells, improves specificity and prevents degradation (more stable).

What does successful miRNA delivery achieve in cancer models?

Blocks proliferation, stops tumour growth, and enhances outcomes when combined with chemo/radiotherapy.

What is stemistry?

The use of small molecules to control stem cell behaviour (proliferation/differentiation/maintainence etc) directly inside the body (in situ).

Why is P7C3 considered an example of stemistry?

P7C3 is a small molecule that enhances survival of endogenous neural progenitor cells by promoting NAD⁺ production. It supports neurogenesis in vivo.

What is MSI-1436 and how does it promote regeneration?

MSI-1436 is a small-molecule inhibitor of protein tyrosine phosphatase 1B. By inhibiting this phosphatase, it enhances signaling pathways involved in heart and skeletal muscle regeneration.

What is the drug name for MSI-1436?

Trodusquemine

What is the proposed mechanism of MSI-1436 action?

It prevents deactivation of phosphorylated receptors by blocking PTP1B and cleavage of phosphate, keeping regenerative pathways switched on.

What is the therapeutic goal of DYRK1A inhibitors in T1DM treatment?

DYRK1A inhibitors aim to promote pancreatic β-cell proliferation by keeping the transcription factor NFAT in the nucleus (prevents the phosphorylation of NFAT), where it drives expression of cell cycle genes.

Why is inhibiting DYRK1A alone not sufficient for effective β-cell regeneration?

It works best in combination with GLP-1 receptor agonists.

What is an example of a DYRK1A inhibitor?

Harmine and INDY

What is the rationale behind using small molecules like AMD3100 for regeneration?

Small molecules can be used to mobilise endogenous stem cells from the bone marrow into the bloodstream, promoting repair and regeneration in damaged tissues.

How does AMD3100 (plerixafor) promote regeneration?

AMD3100 is a CXCR4 antagonist that mobilises BM stem cells into the bloodstream by disrupting the SDF-1/CXCR4 axis, enabling their migration into peripheral tissues for repair (opens the gate)

What is the effect of combining AMD3100 with FK506?

FK506 (tacrolimus) is an immunosuppressant that, in low doses, augments stem cell activation and tissue repair. Combined with AMD3100, it improves cell recruitment and regeneration (e.g., in wound healing and diabetic ulcers).

What was the effect of AMD3100 + tacrolimus in mouse wound healing models?

The combination synergy reduced healing time by 25%, enhanced chemotaxis of stem cells to wound, increased macrophage activation, promoted angiogenesis, and resulted in reduced scarring.

What are quiescent stem cells?

Non-dividing, “dormant” cells that remain inactive until stimulated by injury or stress to re-enter the cell cycle and contribute to tissue repair.

How does aging affect quiescent stem cells?

The stem cell niche changes, leading to fewer cells being able to exit quiescence, reducing tissue repair capacity.

Compare young and old stem cell niches?

Young promotes greater proliferation and migration (Wnt/Ca2+ pathway), whereas old promotes greater quiesence and inflammation.

How might quiescent stem cell activation be achieved therapeutically?

Drugs/small molecules that pharmacologically prime or awaken quiescent stem cells (reversing the cause of senescence) could restore regenerative capacity in aged cells.

What phenotypic changes occur in fibroblasts as they become senescent?

Reduced proliferation and migration, increased cell size and upregulation of AP2A1 which results in a flattened morphology through greater stable focal adhesion with integrins.

What is the function of AP2A1 in senescent fibroblasts?

AP2A1 helps transport integrin β1 along stress fibers, maintaining focal adhesions that increase cell-ECM interaction and contribute to senescent cell enlargement.

What effect does sirNA knockdown of AP2A1 have on senescent cells?

Reverses senescence features: it reduces cell size, increases proliferation, and decreases p21, p53, and SA-β-gal.

What are senolytics?

Senolytics are drugs that selectively destroy senescent cells, allowing surrounding healthy or stem-like cells to proliferate and repair tissue.

What is the goal of rejuvenating stem cells in aging research?

To extend healthspan by reversing intrinsic age-related decline in stem cell function.

What pathway is commonly targeted for intrinsic HSC rejuvenation?

mTOR inhibition by rapamycin (excessive in aging) to prevent stem cells exhaustion and preserve long-term self-renewal.

What organism is commonly studied for true regenerative ability and why?

The axolotl can regenerate entire limbs through dedifferentiation of mature cells into a progenitor state that rebuilds complex tissues, a capacity mammals lack.

Could mammals undergo dedifferentiation for regeneration?

Not naturally. Mammals show partial dedifferentiation, often restricted to certain cell types and are not fully plastic.

What are myoseverin and reversine?

Small molecules to cause partial dedifferentiation of myotubes and induce more plasticity, to reprogram myoblasts into mesenchymal-like progenitors.

What is the Tet-On system used in rejuvenation studies?

A system where doxycycline activates rtTA, which then binds to a Tet operator (tetO) to drive expression of genes like Oct4, Sox2, Klf4, c-Myc (OSKM) in transgenic mice.

What is the purpose of expressing OSKM genes in adult mice?

To partially reprogram somatic cells, promoting cellular rejuvenation without full dedifferentiation into pluripotent stem cells (teratomer risk), resulting in the progressive reversal of aging phenotypes.

What is the effect of long-term partial reprogramming on inflammation and senescence genes in aged mice?

It causes a downregulation of inflammatory and senescence-associated genes, particularly in the skin and kidney.

What are the systemic effects of long-term partial reprogramming in aged mice?

It restores metabolic and lipidomic profiles of the serum to more youthful levels, without altering body weight, survival, or blood counts.

How does long-term partial reprogramming affect tissue plasticity in aged skin?

It leads to a more plastic, less differentiated state in resident cells, making them more responsive to injury and better able to regenerate (increased Ki67 expression).

How is partial reprogramming achieved in aged mice using gene therapy?

By delivering Oct4, Sox2, and Klf4 (OSK) using an AAV vector under a Tet-On system.

What were the key outcomes of gene therapy-mediated partial reprogramming in mice?

109% extension in median remaining lifespan, reduced frailty scores, reversal of epigenetic aging markers in kerinocytes.

What is chemical reprogramming of human cells (hCiPSCs) and why is it challenging?

A method using small molecules instead of transcription factors to induce pluripotency. It's challenging in humans due to a more stable epigenome, which restricts access to DNA and plasticity.

Why is regeneration via dedifferentiation easier in species like axolotls?

Axolotls have open chromatin and naturally maintain a plastic transcriptional state, unlike human cells that need epigenetic resetting for reprogramming.

What changes occur during chemical reprogramming of human fibroblasts into hCiPSCs?

Loss of fibroblast identity, gain of epithelial/pluripotency gene expression, and hypomethylation of pluripotency gene promoters (epigenetic reset).

Loss of fibroblast identity, gain of epithelial/pluripotency gene expression, and hypomethylation of pluripotency gene promoters (epigenetic reset).

Stage I: Loss of fibroblast identity. Stage II: DNA hypomethylation. Stage III: OCT4 activation. Stage IV: Pluripotency network activation

What confirms pluripotency in chemically induced iPSCs (hCiPSCs)?

Teratoma formation and expression of pluripotency markers.

What does the intermediate plastic state resemble in hCiPSCs reprogramming?

The blastema of regenerating axolotl limbs.

What 2 small molecules can be used to reverse the epigenetic ageing of human cells in vitro?

VC6TF and C6NYSA

Why might reprogramming cells not be enough to regenerate tissue like the axolotl?

Reprogramming doesn’t tell cells what to regenerate into or what shape to form. Bioelectricity — electrical fields and ion gradients — provides instructive cues that guide cell growth, shape, and tissue patterning during regeneration.

What is the “bioelectric code” and how does it work alongside the genetic code?

The genetic code (hardware) encodes proteins, but the bioelectric code (software) directs the shape and patterning of tissues. Bioelectric circuits influence cell behaviour tissue patterning.