Compositions and Methods for Delivery of Gene Editing Tools Using Polymeric Vesicles
A composition for genetic modification and a method of forming the composition, the composition may include a synthetic polymer vesicle, and a gene editing system encapsulated in the synthetic polymer vesicle. The gene editing system may include a protein component and a nucleic acid component configured to interact with a target sequence in a host cell genome.
1 . A composition for genetic modification, comprising:
a synthetic polymer vesicle; and
a gene editing system encapsulated in the synthetic polymer vesicle, the gene editing system comprising a nucleic acid component configured to interact with a target sequence in a host cell genome.
2 . The composition of claim 1 , wherein the gene editing system further comprises a protein component.
3 . The composition of claim 2 , wherein:
the protein component comprises a ribonucleic acid (RNA)-directed nuclease; and
the nucleic acid component comprises a guide RNA that is complementary to the target sequence.
4 . The composition of claim 3 , wherein:
the nucleic acid component further comprises an exogenous deoxyribonucleic acid (DNA) repair template;
the RNA-directed nuclease is configured to create a double stranded break in the host cell genome adjacent to the target sequence; and
a repair process in the host cell triggers modification of the host cell genome based on the exogenous DNA repair template, during re-ligation of the host cell genome.
5 . The composition of claim 4 , wherein the DNA repair template comprises end regions that are homologous to regions of the host cell genome flanking the double stranded break induced by the RNA-directed nuclease.
6 . The composition of claim 1 , wherein the gene editing system is included in the synthetic polymer vesicle in an amount of at least 3% by weight relative to the total weight of the composition.
7 . The composition of claim 1 , wherein the protein component comprises an enzyme in native form or a messenger RNA (mRNA) molecule configured to be translated into an enzyme after delivery into the host cell.
8 . The composition of claim 2 , wherein the protein component is delivered as an expression vector containing a deoxyribonucleic acid (DNA) sequence encoding an enzyme.
9 . The composition of claim 8 , wherein the nucleic acid component comprises a DNA sequence encoding a guide ribonucleic acid (RNA).
10 . The composition of claim 9 , wherein the DNA sequence encoding the enzyme and the guide RNA are provided on a single expression vector.
11 . The composition of claim 2 , wherein the protein component comprises an enzyme configured to cut the host genome based on binding of the nucleic acid component to a complementary segment of the host genome.
12 . The composition of claim 11 , wherein the enzyme comprises Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 9 (Cas9).
13 . The composition of claim 2 , wherein:
the nucleic acid component comprises an expression vector that includes a transposon; and
the protein component comprises a transposase.
14 . The composition of claim 13 , wherein the transposase is one of:
a native enzyme;
a messenger ribonucleic acid (mRNA) molecule that is configured to be translated into the enzyme after delivery into the host cell; and
a deoxyribonucleic acid (DNA) sequence encoding the enzyme.
15 . The composition of claim 14 , wherein the DNA sequence is provided on the expression vector that includes the transposon.
16 . The composition of claim 1 , wherein the synthetic polymer vesicle is generated from at least one block copolymer comprising:
a hydrophilic block that includes poly(ethylene oxide); and
a hydrophobic block.
17 . The composition of claim 15 , wherein the hydrophobic block is selected from aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(ortho esters), poly(peptides), poly(phosphazenes) and poly(saccharides).
18 . The composition of claim 15 , wherein the hydrophobic block comprises one or more of poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), or poly (trimethylene carbonate) (PTMC).
19 . A method of modifying a host cell genome, the method comprising:
encapsulating, in a polymersome, a gene editing system that comprises:
a protein component; and
a nucleic acid component configured to interact with a target nucleic acid sequence in the host cell; and
delivering the encapsulated gene editing system to the host cell,
wherein the polymersome is configured to selectively release the gene editing system in the host cell.
20 . The method of claim 19 , wherein delivering the encapsulated gene editing system to the host cell comprises administering to a subject an effective amount of a composition containing the encapsulated gene editing system.
21 . The method of claim 19 , wherein the encapsulated gene editing system is prepared using a progressive saturation protocol.
22 . A method of manufacturing a suspension of an encapsulated gene editing composition, the method comprising:
thermally blending a quantity of a block copolymer with a quantity of a low molecular weight polyethylene glycol (PEG) to create a PEG/polymer formulation;
adding an aliquot of a solution of the gene editing composition to a sample containing the PEG/polymer formulation; and
performing at least one dilution step such that polymersomes that are generated are progressively saturated with the gene editing composition,
wherein the gene editing composition comprises:
a protein component; and
a nucleic acid component configured to interact with a target sequence in a host cell genome.
23 . The method of claim 22 , wherein the block copolymer comprises an amphiphilic diblock copolymer.
24 . The method of claim 23 , wherein the amphiphilic diblock copolymer comprises poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD).
25 . The method of claim 22 , wherein the generated polymersomes have an encapsulation efficiency of at least 50 % with respect to the gene editing composition.
26 . A kit, comprising:
a pharmaceutical composition comprising a gene editing system encapsulated in a synthetic polymer vesicle, the gene editing system comprising:
a protein component; and
a nucleic acid component configured to interact with a target sequence in a host cell genome; and
an implement for administering the pharmaceutical composition intravenously, via inhalation, topically, per rectum, per the vagina, transdermally, subcutaneously, intraperitoneally, intrathecally, intramuscularly, or orally.