Elimination of pro-inflammatory senescent cells has been shown to suppress cancer and rejuvenate tissues by restoring stem cell niches to their healthy state.
Two distinct senolytic approaches will be pursued independently or in combination, to eliminate senescent epithelial cells, associated with pre-cancerous, aging-related lesions:
- Expression of a rapamycin-sensitive allele of the pro-apoptotic protein, Caspase-9 will be restricted to senescent cells using a p16INK4 promoter.
- Expression of a senolytic peptide, derived from FOXO4 shown to disrupt interactions of p53 with FOXO4 in senescence cells (BaarMP17) will be overexpressed from a p16 or constitutive promoter.
¡Transgenes will be encoded by miniaturized plasmids, such as “minicircles” (MCs), and these will be delivered along with permeability enhancers to epithelial cells within a peptide- or polymer-based nanoparticle by topical ointment or biodegradable microneedle patch. Once adjacent to cells, NPs are endocytosed and are designed to rapidly escape from endosomes prior to biodegradation and release of transgene-encoding DNA within the cytosol.
We plan to investigate both transient expression of senolytic proteins and long-term chromatin integration regulated by the senescence-dependent p16 promoter, or an enhanced derivative. [We are also working on a membrane-permeable derivative of the sp that should spread to non-transfected cells.]
Ligand-controlled dimerization of Caspase-9 triggers to its full activation, leading to activation of downstream effector caspases, Caspase-3, -6, and -7) and non-inflammatory apoptosis.
This is a non-immunogenic, low basal activity, cell cycle-independent method of initiating apoptosis and relies on a clinically validated and safe activating ligand
In senescent cells, the p53-interacting protein (p53IP) is upregulated, leading to increased interaction with p53 at the p21 promoter, resulting in increased levels of the cell-cycle inhibitor p21. Enhanced p21 levels not only directly prevent cell proliferation, but its promoter helps to suppress apoptosis, partly by sequestering p53 away from promoters of the pro-apoptotic Bcl-2 family members, Bax and Puma. The p53IP also prevents p53 translocation to the mitochondria where it blocks the functions of anti-apoptotic Bcl-2 and Bcl-2xL.
Expression of a p53-redirecting peptide (p53RP) disrupts the interaction of p53 and p53IP, releasing p53 for re-direction to its alternative pro-apoptotic functions.
DNA plasmid is condensed and packaged by a positively charged peptide (or polymer). DNA- and mRNA (encoding optional transposase)-loaded particles will be formulated for trans- and/or intradermal delivery.
Modular cationic peptide (or polymer) is manufactured separately and engineered to electrostatically interact with oligonucleotide payloads. Amino acid residues selected for optimum endosome escape, DNA/RNA condensation, and rapid degradation in the cytosol.
- Small size (< 100 nm)
- Highly water soluble
- Minimal immunogenicity
There are many options for transdermal DNA delivery, including intradermal injection, electroporation, Gene Gun biolistic particle delivery, and microneedles (MNs), which come in several different forms, such as microchannels, coated, and dissolving MNs, (comprising biodegradable polymers). We plan to use dissolving MNs, due to higher loading payload capacity and ease of use. Although we plan to apply it over a relatively small area, others have shown the practicality and safety of application to a much larger area, including the face in cosmetic applications (before/after shown).
As a lead option, we will load NPs with mini-plasmid along with a tissue permeability-enhancing peptide (PEP) ± transposase mRNA and embed it in a biodegradable Polyvinyl alcohol (PVA) MN mold (shown).
Senescence-Restricted Apoptosis Switch
Activation of the senescence-dependent promoter leads to restricted expression of the pro-apoptotic Rejuvenation Switch in senescent cells. Subsequent administration of the activating ligand triggers safe elimination of these targeted skin-associated senescent cells, releasing stem cell niches from pathological inhibition and resulting in restoration of normal skin rejuvenation and barrier function.
The plasmid backbone can be greatly reduced in size to eliminate antibiotic resistance genes and extraneous foreign DNA to improve expression and facilitate regulatory approval. Alternatively, the therapeutic gene cassette will be stably integrated into the genome using transposon technology, eliminating the bacterial sequence altogether.
Senescence-Restricted Apoptosis-Method 2
Expression of the senolytic peptide within the skin lesion causes apoptosis of senescent cells while leaving unharmed cycling cells and normal stem and precursor cells.
Inclusion of a Scaffold/(Nuclear) Matrix-Attachment Region (S/MAR) ensures prolonged long-term expression of the senolytic peptide.