	US 12,460,230 B2
	Modified mitochondrion and methods of use thereof
Seth Shipman, San Francisco, CA (US)
Assigned to The J. David Gladstone Institutes, San Francisco, CA (US)
Appl. No. 17/429,280
Filed by The J. David Gladstone Institutes, a Testamentary Trust Established Under the Will of J. David Gladstone, San Francisco, CA (US)
PCT Filed Feb. 10, 2020, PCT No. PCT/US2020/017462 § 371(c)(1), (2) Date Aug. 6, 2021, PCT Pub. No. WO2020/163856, PCT Pub. Date Aug. 13, 2020.
Claims priority of provisional application 62/803,546, filed on Feb. 10, 2019.
Prior Publication US 2022/0145330 A1, May 12, 2022
Int. Cl. C12N 15/87 (2006.01); A61K 35/12 (2015.01); A61K 38/16 (2006.01); C07K 14/47 (2006.01)

CPC C12N 15/87 (2013.01) [A61K 35/12 (2013.01); A61K 38/164 (2013.01); C07K 14/47 (2013.01)]

17 Claims

1. An in vitro method of introducing an exogenous modified mitochondrion harvested from a donor cell into a recipient cell, the method comprising:

a. genetically modifying the donor cell to express a fusion protein, the fusion protein comprising:

i. a polypeptide capable of binding to an adhesion molecule on the recipient cell and comprising a portion of an invasin or internalin; and

ii. a polypeptide comprising a mitochondrial outer membrane targeting domain;

b. lysing the donor cell;

c. isolating the exogenous modified mitochondrion from the lysed donor cell of b); and

d. contacting the recipient cell with the isolated exogenous modified mitochondrion from the donor cell of c) and a pore-forming polypeptide, wherein the exogenous modified mitochondrion comprises the fusion protein positioned in the outer membrane of the mitochondrion such that it is capable of binding to the adhesion molecule on the recipient cell; and

wherein the exogenous modified mitochondrion is introduced into the recipient cell,

wherein the donor and recipient cells are mammalian cells.

5. A method of producing a modified mitochondrion, the method comprising genetically modifying a eukaryotic donor cell to express a fusion protein, the fusion protein comprising:

a. a polypeptide capable of binding to an adhesion molecule on a eukaryotic recipient cell; and

b. a polypeptide comprising a mitochondrial outer membrane targeting domain;

wherein the modified mitochondrion is produced and wherein the modified mitochondrion comprises the fusion protein positioned in the outer membrane of the modified mitochondrion such that it is capable of binding to the adhesion molecule on the recipient cell,

wherein the polypeptide capable of binding to the adhesion molecule on the recipient cell comprises an extracellular domain of an invasin or internalin protein.

9. A modified mammalian mitochondrion comprising a fusion protein comprising:

a. a polypeptide comprising an adhesion molecule binding domain; and

b. a polypeptide comprising a mitochondrial outer membrane targeting domain;

wherein the modified mitochondrion comprises the fusion protein positioned in the outer membrane of the modified mitochondrion,

wherein the polypeptide comprising the adhesion molecule domain comprises an extracellular domain of an invasin or internalin protein.

14. A fusion protein, comprising:

a. a polypeptide capable of binding to an adhesion molecule on a recipient cell; and

b. a polypeptide comprising a mitochondrial outer membrane targeting domain,

wherein the polypeptide capable of binding to the adhesion molecule on the recipient cell comprises an extracellular domain of an invasin or internalin protein.