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Encapsulated Bacteria with a Light-Repressed Deadman Switch for Liver Gene Delivery

Submitted:

10 May 2024

Posted:

13 May 2024

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Abstract
Adeno-associated viruses (AAVs) have been used for liver gene therapy. Hemgenix and Roctavian are AAV-based treatments for hemophilia B and A, respectively. They cost $3 million and $2.8 million per dose, respectively. While AAV vectors may eventually be cheaper to mass produce, a bacteria-based DNA delivery system might be much cheaper for patients. Also, “bactofection” would allow for the delivery of much larger DNA packages. Such a bacterial system may now be possible, and a prototype for the liver could possibly be developed immediately.
Keywords: 
bacterial capsule
;  
caged luciferin/luciferase
;  
CY3PA
;  
light-activated allosteric protein switch
;  
Deadman switch
;  
bactofection
Subject: 
Biology and Life Sciences  -   Biology and Biotechnology

Introduction

Adeno-associated viruses (AAVs) can only encode ~5 kb of DNA maximum, which is not sufficient to cure certain individuals with genetic disorders wherein long stretches of nucleotides are affected, e.g., at least some cases of Duchenne muscular dystrophy [1,2,3,4,5,6].
According to Dr. Anzalone et al., “…in principle prime editing can correct up to ~89% of the 75,122 pathogenic human genetic variants in ClinVar…”[7]. New prime editing-based techniques can almost surely cover even more variants[8]. However, multiple proteins/RNAs/DNA molecules (or at least a single, large DNA molecule) may be required in certain cases when more complex molecular machinery must be employed, e.g., if heterochromatin is an issue.
Also, the cost of producing sufficiently high titers of AAV vectors and other viral vectors for therapeutic purposes is very steep[9,10,11].
Lipid nanoparticle (LNP)-encapsulated CRISPR ribonucleoproteins (RNPs) have been successfully utilized for gene delivery and editing in the liver and somewhat in the lungs[12,13]. However, current LNP formulations at least cannot effectively reach or target cells in the central nervous system (CNS), heart, or kidneys after intravenous injection[13,14]. Additionally, the larger the LNP - the less effective it is at extravasation. Thus, it cannot encapsulate large cargos unless targeted to the liver or spleen. Finally, LNP production costs are still substantial, and scaling up the production of new ionizable lipids is not that facile[15,16,17,18].
Intrathecal or intracerebroventricular administration of RNPs, as well as direct intraparenchymal injection of RNPs into the striatum, does not result in widespread CNS editing[19].
A synthetic bacterial vector with low immunogenicity that only delivers to particular organs or organ systems would be of use for multiple reasons:
  • It would be cheap.
  • It could deliver large DNA constructs to use as homologous repair templates or large serine recombinase/CRISPR transposase cargo for individuals with genetic disorders wherein long stretches of nucleotides are affected.
  • May be able to extravasate at least moderately efficiently in most anatomical locales - and then specifically proliferate in the target organ or organ system[20,21]. (If necessary - intraparenchymal injections could be employed.)
  • Flagellar motility of the vector allows for autonomous, widespread delivery throughout the organ or organ system[22].

Prototype for the Liver

Recently, a new system was developed for the programmable encapsulation of microbes to attenuate the immune response and enhance multifocal tumor therapy. An Escherichia Coli Nissle 1917 or a Salmonella Typhimurium vector could be encapsulated to prevent cytotoxic protein secretion from affecting the tissue environment and substantially attenuate the immune response[23]. It can also be myristoylation negative[24]. One could employ an anti-sepsis small molecule, at least in a mouse model[25]. Dexamethasone would be used to further suppress the immune response and induce the expression of CY3PA in the liver. The bacterial vector could then be administered. Finally, one would periodically administer a caged luciferin molecule that is uncaged by CY3PA[26]. The bacterial vector would express firefly luciferase and have a Deadman switch that is sensitive to light[27,28,29,30,31]. Bacteria that stray outside of the liver, where the luciferin is uncaged, would quickly stop replicating and lyse.
When the bacteria in the liver reach a high enough population level (either visualized with an MRI-based reporter gene[32] or just after an experimentally-determined period of time), they can be induced via small molecule to produce a large quantity of CRISPR RNPs[19,33] and lyse.
Theoretically, E. coli Nissle 1917 is endotoxin-free. However, to prevent possible issues from mass lysis, perhaps the small molecule should stop motility immediately - while the gene circuit for lysis can be noisy[34].
Another option would be to use a second Deadman switch; a molecule that the bacteria are exposed to in solution prior to intravenous injection would be gone after injection, and eventually they will just stop replicating and lyse naturally.
Bactofection is a method that has been used for DNA transfer to target cells. It involves invasion of a facultative intracellular bacterium into the a target cell, endosomal rupture, and finally lysis[35]. One of the primary roadblocks to bactofection in vivo has been the immune response to the bacteria.
Notably, only two genes may be required to enable this strategy using E. coli Nissle 1917: the Yersinia pseudotuberculosis invasin and listeriolysin O[36,37].
Having a thick capsule may hinder hepatocyte uptake, however[38]. If so, the bacteria could perhaps divide several times after halting capsule production and overexpressing an eliminase to shed its capsule[39], enter hepatocytes[40], escape the endosome or vacuole[36,37,41], and then lyse.
To increase the efficiency of bactofection, the Listeria monocytogenes actA promoter can be used to drive phage lysin production when the bacteria enter the cytosol of target cells[42]. Using a linear plasmid in combination with NLS-containing proteins that bind said plasmid could substantially increase the efficiency, as linear DNA may be taken up more easily through nuclear pore complexes[43,44]. Moreover, it may be possible to employ a mechanism involving asymmetric division of the vector inside target cells, wherein one of the progeny cells remains in a vacuole and the other lyses to release its cargo[45,46]. The other progeny cell could continue to asymmetrically divide until at least one copy of the DNA construct reaches the host cell nucleus, at which point expression of an artificial gene product would cause it to lyse.
Finally, to strengthen this system, prior to asymmetric division, the bacterium could potentially be allowed to undergo replication up until a tolerable copy number, then being restrained by quorum sensing.
Bacterial entry and release of a replicating RNA vector may also help in the case of RNP delivery, to enhance gene editing efficiency[47,48,49].
For diseases like Hemophilia A and B, not every hepatocyte nucleus would need a copy of the gene construct in order to achieve a curative status, which is helpful with regard to bactofection efficiency and immunogenicity.

Other Organs and Organ Systems

We could identify other enzymes specific to an organ or organ system and develop caged luciferins for those as well. Alternatively, the enzyme could be an organ or organ system-specific extracellular protease, for example. The protease could directly or indirectly activate regulated intramembrane proteolysis[50]. Or, it could cleave a pro-peptide that activates a two-component regulatory system[51]. The benefit of either of those approaches is that an exogenous small molecule would not be required for the continued survival and replication of the vector.
Bacteria can also cross the blood-brain barrier after intravenous injection [21,52].
There is a caged luciferin that can be uncaged in the brain - but it also is uncaged in the kidneys [53]. A better strategy, for now at least, may be to administer a small molecule that is blood-brain barrier-impermeable and that kills any of the synthetic bacteria in the periphery.
In a paper by Dr. Antas et al., it was said that “Although nonviral NPs may tackle the main limitations of their viral counterparts, they have thus far failed to compete with the transduction efficiency achieved by viral vectors in the retina”[54]. Bactofection might be better than AAVs for ocular genetic diseases [55,56]. A Deadman switch could be used to allow limited bacterial replication in the eye via a small molecule present in an eye-drop solution or micro-drug reservoir [57].
Identifying tissue-specific enzymes should not be very difficult nowadays [58].

Conclusions

Encapsulated bacteria with a light-repressed Deadman switch could be used for liver gene delivery. They may be much cheaper than AAV-based liver gene therapies, and even LNPs. It would also allow for the delivery of large DNA packages. In the future, other organs that are harder to reach with gene therapy or editing components, like the heart or brain, could be treated with this therapy.

References

  1. Spinner NB, Loomes KM, Krantz ID, Gilbert MA. Alagille Syndrome. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Bean LJ, et al., editors. GeneReviews®, Seattle (WA): University of Washington, Seattle; 1993.
  2. Stoller JK, Hupertz V, Aboussouan LS. Alpha-1 Antitrypsin Deficiency. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Bean LJ, et al., editors. GeneReviews®, Seattle (WA): University of Washington, Seattle; 1993.
  3. Myerowitz, R. Tay-Sachs disease-causing mutations and neutral polymorphisms in the Hex A gene. Hum Mutat 1997;9:195–208. [CrossRef]
  4. Butchbach, M.E.R. Genomic Variability in the Survival Motor Neuron Genes (SMN1 and SMN2): Implications for Spinal Muscular Atrophy Phenotype and Therapeutics Development. Int. J. Mol. Sci. 2021, 22, 7896. [Google Scholar] [CrossRef] [PubMed]
  5. Daiger, S.P.; Sullivan, L.S.; Bowne, S.J. Genes and mutations causing retinitis pigmentosa. Clin. Genet. 2013, 84, 132–141. [Google Scholar] [CrossRef] [PubMed]
  6. Duan, D.; Goemans, N.; Takeda, S.; Mercuri, E.; Aartsma-Rus, A. Duchenne muscular dystrophy. Nat. Rev. Dis. Primers 2021, 7, 13. [Google Scholar] [CrossRef] [PubMed]
  7. Anzalone, A.V.; Randolph, P.B.; Davis, J.R.; Sousa, A.A.; Koblan, L.W.; Levy, J.M.; Chen, P.J.; Wilson, C.; Newby, G.A.; Raguram, A.; et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019, 576, 149–157. [Google Scholar] [CrossRef] [PubMed]
  8. Zheng, C.; Liu, B.; Dong, X.; Gaston, N.; Sontheimer, E.J.; Xue, W. Template-jumping prime editing enables large insertion and exon rewriting in vivo. Nat. Commun. 2023, 14, 1–9. [Google Scholar] [CrossRef] [PubMed]
  9. Collins, L.T.; Ponnazhagan, S.; Curiel, D.T. Synthetic Biology Design as a Paradigm Shift toward Manufacturing Affordable Adeno-Associated Virus Gene Therapies. ACS Synth. Biol. 2023, 12, 17–26. [Google Scholar] [CrossRef] [PubMed]
  10. Becker, Z. Sporting a $3.5M price tag, CSL and uniQure’s hemophilia B gene therapy crosses FDA finish line. Fierce Pharma 2022. https://www.fiercepharma.com/pharma/csl-and-uniqures-hemophilia-b-gene-therapy-scores-approval-35-million-price-tag (accessed , 2023). 9 December.
  11. Bluebird Bio Secures Deal with Large Commercial Payer for Lyfgenia Amid Price Concerns. BioSpace n.d. https://www.biospace.com/article/bluebird-bio-secures-deal-with-large-commercial-payer-for-lyfgenia-amid-price-concerns/ (accessed , 2024). 4 January.
  12. Wei, T.; Sun, Y.; Cheng, Q.; Chatterjee, S.; Traylor, Z.; Johnson, L.T.; Coquelin, M.L.; Wang, J.; Torres, M.J.; Lian, X.; et al. Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models. Nat. Commun. 2023, 14, 1–14. [Google Scholar] [CrossRef] [PubMed]
  13. Chen K, Han H, Zhao S, Xu B, Yin B, Trinidad M, et al. Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 RNP 2023:2023.11.15.566339. [CrossRef]
  14. Behr, M.; Zhou, J.; Xu, B.; Zhang, H. In vivo delivery of CRISPR-Cas9 therapeutics: Progress and challenges. Acta Pharm. Sin. B 2021, 11, 2150–2171. [Google Scholar] [CrossRef] [PubMed]
  15. Wang, X.; Liu, S.; Sun, Y.; Yu, X.; Lee, S.M.; Cheng, Q.; Wei, T.; Gong, J.; Robinson, J.; Zhang, D.; et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat. Protoc. 2022, 18, 265–291. [Google Scholar] [CrossRef]
  16. Verma, M.; Ozer, I.; Xie, W.; Gallagher, R.; Teixeira, A.; Choy, M. The landscape for lipid-nanoparticle-based genomic medicines. Nat. Rev. Drug Discov. 2023, 22, 349–350. [Google Scholar] [CrossRef]
  17. Shepherd, S.J.; Han, X.; Mukalel, A.J.; El-Mayta, R.; Thatte, A.S.; Wu, J.; Padilla, M.S.; Alameh, M.-G.; Srikumar, N.; Lee, D.; et al. Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines. Proc. Natl. Acad. Sci. 2023, 120. [Google Scholar] [CrossRef]
  18. Mehta, M.; Bui, T.A.; Yang, X.; Aksoy, Y.; Goldys, E.M.; Deng, W. Lipid-Based Nanoparticles for Drug/Gene Delivery: An Overview of the Production Techniques and Difficulties Encountered in Their Industrial Development. ACS Mater. Au 2023, 3, 600–619. [Google Scholar] [CrossRef]
  19. Stahl, E.C.; Sabo, J.K.; Kang, M.H.; Allen, R.; Applegate, E.; Kim, S.E.; Kwon, Y.; Seth, A.; Lemus, N.; Salinas-Rios, V.; et al. Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs. Mol. Ther. 2023, 31, 2422–2438. [Google Scholar] [CrossRef]
  20. Tan, X.; Petri, B.; DeVinney, R.; Jenne, C.N.; Chaconas, G. The Lyme disease spirochete can hijack the host immune system for extravasation from the microvasculature. Mol. Microbiol. 2021, 116, 498–515. [Google Scholar] [CrossRef] [PubMed]
  21. Sun, R.; Liu, M.; Lu, J.; Chu, B.; Yang, Y.; Song, B.; Wang, H.; He, Y. Bacteria loaded with glucose polymer and photosensitive ICG silicon-nanoparticles for glioblastoma photothermal immunotherapy. Nat. Commun. 2022, 13, 1–15. [Google Scholar] [CrossRef] [PubMed]
  22. Toley, B.J.; Forbes, N.S. Motility is critical for effective distribution and accumulation of bacteria in tumor tissue. Integr. Biol. 2011, 4, 165–176. [Google Scholar] [CrossRef] [PubMed]
  23. Harimoto, T.; Hahn, J.; Chen, Y.-Y.; Im, J.; Zhang, J.; Hou, N.; Li, F.; Coker, C.; Gray, K.; Harr, N.; et al. A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Nat. Biotechnol. 2022, 40, 1259–1269. [Google Scholar] [CrossRef] [PubMed]
  24. Stritzker, J.; Hill, P.J.; Gentsche, I.; Szalay, A.A. Myristoylation negative msbB-mutants of probiotic E. coli Nissle 1917 retain tumor specific colonization properties but show less side effects in immunocompetent mice. Bioeng. Bugs 2010, 1, 139–145. [Google Scholar] [CrossRef] [PubMed]
  25. Gawish, R.; Maier, B.; Obermayer, G.; Watzenboeck, M.L.; Gorki, A.-D.; Quattrone, F.; Farhat, A.; Lakovits, K.; Hladik, A.; Korosec, A.; et al. A neutrophil–B-cell axis impacts tissue damage control in a mouse model of intraabdominal bacterial infection via Cxcr4. eLife 2022, 11. [Google Scholar] [CrossRef] [PubMed]
  26. Yevtodiyenko, A.; Bazhin, A.; Khodakivskyi, P.; Godinat, A.; Budin, G.; Maric, T.; Pietramaggiori, G.; Scherer, S.S.; Kunchulia, M.; Eppeldauer, G.; et al. Portable bioluminescent platform for in vivo monitoring of biological processes in non-transgenic animals. Nat. Commun. 2021, 12, 1–12. [Google Scholar] [CrossRef]
  27. Mi, Z.; Yao, Q.; Qi, Y.; Zheng, J.; Liu, J.; Liu, Z.; Tan, H.; Ma, X.; Zhou, W.; Rong, P. Salmonella-mediated blood‒brain barrier penetration, tumor homing and tumor microenvironment regulation for enhanced chemo/bacterial glioma therapy. Acta Pharm. Sin. B 2023, 13, 819–833. [Google Scholar] [CrossRef]
  28. Dash, R.; Holsinger, K.A.; Chordia, M.D.; Gh. , M.S.; Pires, M.M. Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli. ACS Infect. Dis. 2024, 10, 1602–1611. [Google Scholar] [CrossRef]
  29. Chan, C.T.Y.; Lee, J.W.; Cameron, D.E.; Bashor, C.J.; Collins, J.J. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat. Chem. Biol. 2015, 12, 82–86. [Google Scholar] [CrossRef]
  30. Branchini, B.R.; Ablamsky, D.M.; Rosenman, J.M.; Uzasci, L.; Southworth, T.L.; Zimmer, M. Synergistic Mutations Produce Blue-Shifted Bioluminescence in Firefly Luciferase. Biochemistry 2007, 46, 13847–13855. [Google Scholar] [CrossRef]
  31. Nash, A.I.; McNulty, R.; Shillito, M.E.; Swartz, T.E.; Bogomolni, R.A.; Luecke, H.; Gardner, K.H. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proc. Natl. Acad. Sci. 2011, 108, 9449–9454. [Google Scholar] [CrossRef]
  32. Allouche-Arnon, H.; Khersonsky, O.; Tirukoti, N.D.; Peleg, Y.; Dym, O.; Albeck, S.; Brandis, A.; Mehlman, T.; Avram, L.; Harris, T.; et al. Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression. Nat. Biotechnol. 2022, 40, 1143–1149. [Google Scholar] [CrossRef]
  33. Yu, S.; Yang, H.; Li, T.; Pan, H.; Ren, S.; Luo, G.; Jiang, J.; Yu, L.; Chen, B.; Zhang, Y.; et al. Efficient intracellular delivery of proteins by a multifunctional chimaeric peptide in vitro and in vivo. Nat. Commun. 2021, 12, 1–13. [Google Scholar] [CrossRef]
  34. Kannoly, S.; Gao, T.; Dey, S.; Wang, I.-N.; Singh, A.; Dennehy, J.J. Optimum Threshold Minimizes Noise in Timing of Intracellular Events. iScience 2020, 23, 101186. [Google Scholar] [CrossRef]
  35. Sultana, A.; Kumar, R. Modified bactofection for efficient and functional DNA delivery using invasive E. coli DH10B vector into human epithelial cell line. J. Drug Deliv. Sci. Technol. 2022, 70, 103159. [Google Scholar] [CrossRef]
  36. Grillot-Courvalin, C.; Goussard, S.; Huetz, F.; Ojcius, D.M.; Courvalin, P. Functional gene transfer from intracellular bacteria to mammalian cells. Nat. Biotechnol. 1998, 16, 862–866. [Google Scholar] [CrossRef]
  37. Zare, M.; Farhadi, A.; Zare, F.; Dehbidi, G.R.; Zarghampoor, F.; Ahmadi, M.K.B.; Behbahani, A.B. Genetically engineered E. coli invade epithelial cells and transfer their genetic cargo into the cells: an approach to a gene delivery system. Biotechnol. Lett. 2023, 45, 861–871. [Google Scholar] [CrossRef] [PubMed]
  38. Schembri, M.A.; Dalsgaard, D.; Klemm, P. Capsule Shields the Function of Short Bacterial Adhesins. J. Bacteriol. 2004, 186, 1249–57. [Google Scholar] [CrossRef]
  39. Hickey, A.M.; Bhaskar, U.; Linhardt, R.J.; Dordick, J.S. Effect of eliminase gene (elmA) deletion on heparosan production and shedding in Escherichia coli K5. J. Biotechnol. 2013, 165, 175–177. [Google Scholar] [CrossRef]
  40. Raman, V.; Van Dessel, N.; Hall, C.L.; Wetherby, V.E.; Whitney, S.A.; Kolewe, E.L.; Bloom, S.M.K.; Sharma, A.; Hardy, J.A.; Bollen, M.; et al. Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases. Nat. Commun. 2021, 12, 1–14. [Google Scholar] [CrossRef] [PubMed]
  41. Brumell, J.H.; Tang, P.; Zaharik, M.L.; Finlay, B.B. Disruption of the Salmonella-Containing Vacuole Leads to Increased Replication of Salmonella enterica Serovar Typhimurium in the Cytosol of Epithelial Cells. Infect. Immun. 2002, 70, 3264–3270. [Google Scholar] [CrossRef] [PubMed]
  42. Pilgrim, S.; Stritzker, J.; Schoen, C.; Kolb-Mäurer, A.; Geginat, G.; Loessner, M.J.; Gentschev, I.; Goebel, W. Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery. Gene Ther. 2003, 10, 2036–2045. [Google Scholar] [CrossRef] [PubMed]
  43. Johansson, P.; Lindgren, T.; Lundström, M.; Holmström, A.; Elgh, F.; Bucht, G. PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine. Vaccine 2002, 20, 3379–3388. [Google Scholar] [CrossRef] [PubMed]
  44. Zhu, J.; Batra, H.; Ananthaswamy, N.; Mahalingam, M.; Tao, P.; Wu, X.; Guo, W.; Fokine, A.; Rao, V.B. Design of bacteriophage T4-based artificial viral vectors for human genome remodeling. Nat. Commun. 2023, 14, 2928. [Google Scholar] [CrossRef] [PubMed]
  45. Molinari, S.; Shis, D.L.; Bhakta, S.P.; Chappell, J.; Igoshin, O.A.; Bennett, M.R. A synthetic system for asymmetric cell division in Escherichia coli. Nat. Chem. Biol. 2019, 15, 917–924. [Google Scholar] [CrossRef]
  46. Eswarappa, S.M.; Negi, V.D.; Chakraborty, S.; Sagar, B.K.C.; Chakravortty, D. Division of theSalmonella-Containing Vacuole and Depletion of Acidic Lysosomes inSalmonella-Infected Host Cells Are Novel Strategies ofSalmonella entericaTo Avoid Lysosomes. Infect. Immun. 2010, 78, 68–79. [Google Scholar] [CrossRef]
  47. Pabon, J.; Singer, Z.; Huang, H.; Rice, C.; Danino, T. Abstract 6906: Engineered bacteria launch and control an oncolytic virus. Cancer Res 2024, 84, 6906–6906. [Google Scholar] [CrossRef]
  48. Mc Cafferty, S.; De Temmerman, J.; Kitada, T.; Becraft, J.R.; Weiss, R.; Irvine, D.J.; Devreese, M.; De Baere, S.; Combes, F.; Sanders, N.N. In Vivo Validation of a Reversible Small Molecule-Based Switch for Synthetic Self-Amplifying mRNA Regulation. Mol. Ther. 2020, 29, 1164–1173. [Google Scholar] [CrossRef] [PubMed]
  49. Perkovic, M.; Gawletta, S.; Hempel, T.; Brill, S.; Nett, E.; Sahin, U.; Beissert, T. A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in mice. Mol. Ther. 2023, 31, 1636–1646. [Google Scholar] [CrossRef]
  50. Heinrich, J.; Wiegert, T. Regulated intramembrane proteolysis in the control of extracytoplasmic function sigma factors. Res. Microbiol. 2009, 160, 696–703. [Google Scholar] [CrossRef] [PubMed]
  51. Brink, K.R.; Hunt, M.G.; Mu, A.M.; Groszman, K.; Hoang, K.V.; Lorch, K.P.; Pogostin, B.H.; Gunn, J.S.; Tabor, J.J. An E. coli display method for characterization of peptide–sensor kinase interactions. Nat. Chem. Biol. 2022, 19, 451–459. [Google Scholar] [CrossRef] [PubMed]
  52. Arbab, M.; Matuszek, Z.; Kray, K.M.; Du, A.; Newby, G.A.; Blatnik, A.J.; Raguram, A.; Richter, M.F.; Zhao, K.T.; Levy, J.M.; et al. Base editing rescue of spinal muscular atrophy in cells and in mice. Science 2023, 380, eadg6518–eadg6518. [Google Scholar] [CrossRef] [PubMed]
  53. Mofford, D.M.; Adams, S.T.; Reddy, G.S.K.K.; Miller, S.C. Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity. J. Am. Chem. Soc. 2015, 137, 8684–8687. [Google Scholar] [CrossRef]
  54. Antas, P.; Carvalho, C.; Cabral-Teixeira, J.; de Lemos, L.; Seabra, M.C. Toward low-cost gene therapy: mRNA-based therapeutics for treatment of inherited retinal diseases. Trends Mol. Med. 2024, 30, 136–146. [Google Scholar] [CrossRef] [PubMed]
  55. Yiu, G.; Chung, S.H.; Mollhoff, I.N.; Nguyen, U.T.; Thomasy, S.M.; Yoo, J.; Taraborelli, D.; Noronha, G. Suprachoroidal and Subretinal Injections of AAV Using Transscleral Microneedles for Retinal Gene Delivery in Nonhuman Primates. Mol. Ther. - Methods Clin. Dev. 2020, 16, 179–191. [Google Scholar] [CrossRef]
  56. Chuah, J.-A.; Matsugami, A.; Hayashi, F.; Numata, K. Self-Assembled Peptide-Based System for Mitochondrial-Targeted Gene Delivery: Functional and Structural Insights. Biomacromolecules 2016, 17, 3547–3557. [Google Scholar] [CrossRef]
  57. Than, A.; Liu, C.; Chang, H.; Duong, P.K.; Cheung, C.M.G.; Xu, C.; Wang, X.; Chen, P. Self-implantable double-layered micro-drug-reservoirs for efficient and controlled ocular drug delivery. Nat. Commun. 2018, 9, 1–12. [Google Scholar] [CrossRef] [PubMed]
  58. Yang RY, Quan J, Sodaei R, Aguet F, Segrè AV, Allen JA, et al. A systematic survey of human tissue-specific gene expression and splicing reveals new opportunities for therapeutic target identification and evaluation 2018:311563. [CrossRef]
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