Periodic Reporting for period 1 - HCMSpliceAOC (Molecular design and application of splice-switching antibody-oligonucleotide conjugate for hypertrophic cardiomyopathy-related MYBPC3 splice modulation.)
Okres sprawozdawczy: 2023-06-01 do 2025-05-31
The delivery of oligonucleotides, short polymeric nucleotide sequences of nucleic acids, into the site of action in the cytosol or nucleus of cells remains to be a key challenge in oligonucleotide-based therapeutics. It is indeed estimated that only approximately 1.5-3% of short interfering RNAs (siRNAs) comprised of duplex oligonucleotides reach the cytoplasm after the occurrence of an endosomal escape event (Gilleron, J. et al. Nat. Biotechnol., 2013). Consequently, longer nucleotide sequences in oligonucleotides and mRNA-based vaccines are even more difficult to deliver. To date, no antibody- or protein-oligonucleotide conjugates have reached the drug market. We herein propose a bioconjugation strategy (Figure 1) consitsting of an oligonucleotide conjugated to both a protease-cleavable linker and a receptor-binding mini protein that can potentially promote rapid uptake and endosomal disruption in cells. Briefly, the oligonucleotide conjugate will contain modifiers on both ends to conjugate traceless linkers to enable cleavage by cathepsin B, naturally present in cells. Furthermore, we will employ a quaternary ammonium to give the conjugate a positive charge and reduce the likelihood of aggregation. This project is in collaborative with both Prof. David Baker’s lab at the Institute of Protein Design who has published work on de novo design of human TfR1 receptor binders, and Prof. Maria Carmo-Fonseca’s lab at Instituto de Medicina Molecular who has expertise in RNA splicing and splice-switching oligonucleotide-based therapeutic development. As proof-of-concept, we aimed to design and test an ASO-protein-linker conjugate that could target the tumor suppressor Menin1 (MEN1) gene in breast cancer cells. We expect that this project can deliver solutions to facilitate improved oligonucleotide therapeutic delivery in the industrial market.
The project involved the following activities:
1) Conjugate fluorophore to TfR1 mini binder protein;
2) Confirm internalization of TfR1 mini binder in cells;
3) Generate stably expressing mCherry-Gal9+ cells to quantify endosomal escape of ASO-protein conjugate;
4) Synthesize the valine-citrulline linker bearing a propargyl quaternay ammonium modification;
5) Conjugate linker to control azide lipid or oligonucleotide;
6) Test and quantify endosomal escape of ASO-protein conjugate in cells
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1) Conjugate fluorophore to TfR1 mini binder protein:
A ~25 kDa mini protein derived from work by Sahtoe and colleagues at the Institute for Protein Design (Sahtoe, D.D. et. al., PNAS, 2021) were sent to us for conjugation to fluorophores and subsequent testing for internalization in cells. This protein binds with high (nanomolar) affinity to the transferring receptor 1 (TfR1) and contains a single cysteine mutant for conjugation to fluorophores bearing a maleimide moiety via thiol-maleimide chemistry.
Outcome: A variety of fluorophores were tested and successfully conjugated to the protein.
2) Confirm internalization of mini binder in cells:
The fluorophore-protein conjugated was tested for internalization in breast cancer cell lines Hs578t and T47d, both of which are reported to contain sufficient levels of TfR1 and MEN1 for ASO endocytosis and oligonuclotide activity, respectively (Nguyen, D.T. et. al., Biomedicines, 2021).
Outcome: TfR1 mini binder conjugated to fluorescein, Cyanine3, or Cyanine5 were successfully internalized in Hs578t and T47d cells.
3) Generate stably expressing mCherry-Gal9+ cells to quantify endosomal escape of ASO-protein conjugate:
To quantify endosomal escape, we adapted a reporter system from Munson and colleagues by generating stably expressed mCherry-Gal9+, a fusion fluorescent protein that forms punctate spots at sites of endosomal disruption sites which can be observed by fluorescence microscopy (Munson, M.J. et. al., Nat. Comm. Bio., 2021). To do this, we co-transfected mCherry-Gal9+ reporter and with genomic integration Zinc Finger Nuclease expression (pCMV-AAVS-ZFN) plasmids in cells and selected for mCherry+ cells by puromycin antibiotic selection followed by cell sorting.
Outcome: T-47d and control HEK293T cells were successfully transfected and selected for stable generation of mCherry-Gal9+.
4) Synthesize the valine-citrulline linker bearing a propargyl quaternary ammonium modification:
The synthetic scheme to make the modified protease-cleavable linker and subsequent conjugation to TfR1 mini binder protein and to ASO are outlined in Scheme 1. In brief, a protease-cleavable valine-citrulline para-aminobenzyl alcohol (1) is concerted into a chloride (2) by addition of thionyl chloride (SOCl2). Next, the chloride is converted into a propargyl quaternary ammonium moiety (3) by treatment with 3-dimethylamino-1-propyne, tetrabutyl ammonium iodide (TBAI), and Hunig's base. The linker is then conjugated to the TfR1 mini binder (4) by a simple conjugation reaction in 10% DMF and Tris-buffer for approximately 2 hours. Finally, the protein-linker conjugate is furthermore conjugated to azido-modified MEN1 ASO via copper(I)-catalyzed click chemistry reaction (5).
Outcome: We successfully generated molecule 4 and are working on conjugating to ASO.
5) Conjugate linker to control azide lipid or oligonucleotide:
Outcome: We have successfully generated molecule 4 and conjugated the protein-linker to a control myristic acid (Scheme 2, molceule 6) that has structural similarity to the azido-modified ASO, provided by commercial sources (Integrated DNA Technologies, IDT). Conjugation of protein-linker to ASO is in progress.
6) Test and quantify endosomal escape of ASO-protein conjugate in cells:
Outcome: This stage of the project was not achieved, but we anticipate that conjugation of the protein-linker to ASO would be simple and could therefore be easily tested for endosomal escape.
1) Conjugate fluorophore to TfR1 mini binder protein;
2) Confirm internalization of TfR1 mini binder in cells;
3) Generate stably expressing mCherry-Gal9+ cells to quantify endosomal escape of ASO-protein conjugate;
4) Synthesize the valine-citrulline linker bearing a propargyl quaternay ammonium modification;
5) Conjugate linker to control azide lipid or oligonucleotide;
6) Test and quantify endosomal escape of ASO-protein conjugate in cells
---
1) Conjugate fluorophore to TfR1 mini binder protein:
A ~25 kDa mini protein derived from work by Sahtoe and colleagues at the Institute for Protein Design (Sahtoe, D.D. et. al., PNAS, 2021) were sent to us for conjugation to fluorophores and subsequent testing for internalization in cells. This protein binds with high (nanomolar) affinity to the transferring receptor 1 (TfR1) and contains a single cysteine mutant for conjugation to fluorophores bearing a maleimide moiety via thiol-maleimide chemistry.
Outcome: A variety of fluorophores were tested and successfully conjugated to the protein.
2) Confirm internalization of mini binder in cells:
The fluorophore-protein conjugated was tested for internalization in breast cancer cell lines Hs578t and T47d, both of which are reported to contain sufficient levels of TfR1 and MEN1 for ASO endocytosis and oligonuclotide activity, respectively (Nguyen, D.T. et. al., Biomedicines, 2021).
Outcome: TfR1 mini binder conjugated to fluorescein, Cyanine3, or Cyanine5 were successfully internalized in Hs578t and T47d cells.
3) Generate stably expressing mCherry-Gal9+ cells to quantify endosomal escape of ASO-protein conjugate:
To quantify endosomal escape, we adapted a reporter system from Munson and colleagues by generating stably expressed mCherry-Gal9+, a fusion fluorescent protein that forms punctate spots at sites of endosomal disruption sites which can be observed by fluorescence microscopy (Munson, M.J. et. al., Nat. Comm. Bio., 2021). To do this, we co-transfected mCherry-Gal9+ reporter and with genomic integration Zinc Finger Nuclease expression (pCMV-AAVS-ZFN) plasmids in cells and selected for mCherry+ cells by puromycin antibiotic selection followed by cell sorting.
Outcome: T-47d and control HEK293T cells were successfully transfected and selected for stable generation of mCherry-Gal9+.
4) Synthesize the valine-citrulline linker bearing a propargyl quaternary ammonium modification:
The synthetic scheme to make the modified protease-cleavable linker and subsequent conjugation to TfR1 mini binder protein and to ASO are outlined in Scheme 1. In brief, a protease-cleavable valine-citrulline para-aminobenzyl alcohol (1) is concerted into a chloride (2) by addition of thionyl chloride (SOCl2). Next, the chloride is converted into a propargyl quaternary ammonium moiety (3) by treatment with 3-dimethylamino-1-propyne, tetrabutyl ammonium iodide (TBAI), and Hunig's base. The linker is then conjugated to the TfR1 mini binder (4) by a simple conjugation reaction in 10% DMF and Tris-buffer for approximately 2 hours. Finally, the protein-linker conjugate is furthermore conjugated to azido-modified MEN1 ASO via copper(I)-catalyzed click chemistry reaction (5).
Outcome: We successfully generated molecule 4 and are working on conjugating to ASO.
5) Conjugate linker to control azide lipid or oligonucleotide:
Outcome: We have successfully generated molecule 4 and conjugated the protein-linker to a control myristic acid (Scheme 2, molceule 6) that has structural similarity to the azido-modified ASO, provided by commercial sources (Integrated DNA Technologies, IDT). Conjugation of protein-linker to ASO is in progress.
6) Test and quantify endosomal escape of ASO-protein conjugate in cells:
Outcome: This stage of the project was not achieved, but we anticipate that conjugation of the protein-linker to ASO would be simple and could therefore be easily tested for endosomal escape.
This project has made some progress. More research would need to be performed to generate results worthy of publication and to demonstrate the proof-of-concept of enhanced intracellular delivery and endosomal escape of our ASO-protein conjugate construct.
To summarize, we successfully confirmed rapid internalization of fluorophore-conjugated TfR1 mini binder protein in breast cancer Hs578t and T47d cells (Figure 2a-c). T47d and HEK293T stably expressing mCherry-Gal9 were generated (Figure 3). Finally, the quaternary ammonium-modified conjugate consisting of mini binder protein TfR1, HER2, or PDL1, valine-citrulline linker, and myristic acid at 80% or 100% efficiency, as determined by mass spectrometry (Figure 4).
This project should be easily carried over to conjugation of protein-linker to oligonucleotide for quantifying endosomal escape and MEN1 silencing activity.
To summarize, we successfully confirmed rapid internalization of fluorophore-conjugated TfR1 mini binder protein in breast cancer Hs578t and T47d cells (Figure 2a-c). T47d and HEK293T stably expressing mCherry-Gal9 were generated (Figure 3). Finally, the quaternary ammonium-modified conjugate consisting of mini binder protein TfR1, HER2, or PDL1, valine-citrulline linker, and myristic acid at 80% or 100% efficiency, as determined by mass spectrometry (Figure 4).
This project should be easily carried over to conjugation of protein-linker to oligonucleotide for quantifying endosomal escape and MEN1 silencing activity.