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US 11,692,196 B2Granted
Methods for creating synthetic chromosomes expressing biosynthetic pathways and uses thereof
AssigneeCarryGenes Bioengineering, LLC
InventorsEdward Perkins, Amy Greene
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Abstract
The present invention encompasses compositions and methods to allow one to deliver and express multiple genes from a biosynthetic pathway in a recipient cell via a synthetic chromosome.
Claims12
A method for constructing a mammalian synthetic chromosome to express genes encoding enzymes involved in the synthesis of metabolites in a biosynthetic pathway in a host cell comprising:
- (a) transfecting a satellite DNA-based mammalian synthetic chromosome production cell line with synthetic chromosome production components comprising nucleic acids engineered to incorporate multiple site-specific integration sites into a satellite DNA-based synthetic platform chromosome as it is produced in the satellite DNA-based mammalian synthetic chromosome production cell line;
- (b) identifying and isolating an engineered mammalian cell carrying the satellite DNA-based mammalian synthetic platform chromosome comprising multiple site-specific integration sites produced in (a);
- (c) transfecting the isolated engineered mammalian cell of (b) carrying the synthetic platform chromosome with a delivery vector comprising at least one site-specific recombination site and multiple genes encoding multiple gene products in a first biosynthetic pathway, wherein multiple site-specific recombination sites and integration sites are selected from attachment site pairs comprising target DNA sequences attB/attP and attL/attR, or mutated versions of attP/attB and attL/attR;
- (d) activating site-specific recombination between the synthetic platform chromosome and the delivery vector using an integrase, wherein the multiple genes encoding multiple gene products in the first biosynthetic pathway are loaded onto the synthetic platform chromosome in the engineered cell to produce a satellite DNA-based mammalian synthetic chromosome expressing multiple gene products that act in concert in the first biosynthetic pathway;
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- (e) isolating a first host cell comprising the satellite DNA-based mammalian synthetic chromosome, wherein the first host cell expresses the multiple gene products that act in concert to produce metabolites in the first biosynthetic pathway.
3 more elements
A method for constructing a mammalian synthetic chromosome to express genes encoding enzymes involved in the synthesis of metabolites in a biosynthetic pathway in a host immune cell comprising:
- (a) transfecting a satellite DNA-based mammalian synthetic chromosome production cell line with synthetic chromosome production components comprising nucleic acids engineered to incorporate multiple site-specific integration sites into a satellite DNA-based synthetic platform chromosome as it is produced in the satellite DNA-based mammalian synthetic chromosome production cell line;
- (b) identifying and isolating an engineered mammalian cell carrying the satellite DNA-based mammalian synthetic platform chromosome comprising multiple site-specific integration sites produced in (a);
- (c) transfecting the isolated engineered mammalian cell of (b) carrying the synthetic platform chromosome with a delivery vector comprising at least one site-specific recombination site and multiple genes encoding multiple gene products in a first biosynthetic pathway, wherein multiple site-specific recombination sites and integration sites are selected from attachment site pairs comprising target DNA sequences attB/attP and attL/attR, or mutated versions of attP/attB and attL/attR;
- (d) activating site-specific recombination between the satellite DNA-based synthetic platform chromosome and the delivery vector using an integrase, wherein the multiple genes encoding multiple gene products in the first biosynthetic pathway are loaded onto the synthetic platform chromosome in the engineered cell to produce a satellite DNA-based mammalian synthetic chromosome expressing multiple gene products that act in concert in the first biosynthetic pathway;
- and
- (e) isolating a first host cell comprising the satellite DNA-based mammalian synthetic chromosome, wherein the first host cell expresses the multiple gene products in the first biosynthetic pathway;
- (f) isolating the satellite DNA-based mammalian synthetic chromosome expressing the multiple gene products that act in concert to produce metabolites in the first biosynthetic pathway;
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- (g) transferring the satellite DNA-based mammalian synthetic chromosome to a recipient host immune cell.
6 more elements
Dependencies are inferred from the claim text.