Treating diabetes with genetically modified beta cells

Inactive Publication Date: 2019-04-04
SDF BIOPHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]In some embodiments, the fugetactic agent is a modified fugetactic agent. For example, the polypeptide sequence of the fugetactic agent may be modified to increase circulating half-life, to incorporate conservative amino acid changes, enhance binding to an extracellular matrix, improve activity of the agent, etc. Accordingly, genes encoding a modified f

Problems solved by technology

In subjects with type 1 diabetes (T1D), beta cells are attacked and destroyed by the immune system and, as a result, subjects with T1D cannot efficiently produce their own insulin.
Type 2 diabetes (T2D) occurs when a subject's persistently high blood sugar overwhelms the capacity of a subject's beta-cells to produce enough insulin to prevent hyperglycemia in the subject and leads to beta-cell malfunction, de-differentiation, and death.
However, infiltration of mononuclear immune cells (T-cells, B-cells, and NK cells) results in the failure of the beta cell transplantatio

Method used

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  • Treating diabetes with genetically modified beta cells
  • Treating diabetes with genetically modified beta cells
  • Treating diabetes with genetically modified beta cells

Examples

Experimental program
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Effect test

example 1

ls Used to Assess Expression Levels of CXCL12-a and -b Isoforms

[0119]HEK293 Cells were Transfected with 2 Different Isoforms of CXCL12 (Alpha and Beta) using commercially available plasmids for each isoform (plasmids available from GenScript). Transfected cells were selected with 250 ug / mL of G418 (commercially available from ThermoFisher) and a stable pool for each isoform was created. Cells were allowed to condition a suitable medium for 3 days. Conditioned medium from the transfected HEK293 cells expressing CXCL12 alpha and CXCL12 beta were diluted 1:1 with assay dilution buffer. Two separate pools were established for each isoform and then the concentration of each isoform in solution were obtained by absorption using a standardized concentration curve.

[0120]This experiment was repeated twice and the results are as follows:

CXCL12 alphaCXCL12 beta1.310 nM1410 nM2.274 nM1330 nM

[0121]The above results evidence that transgenic model cells express CXCL12 beta at significantly higher ...

example 2

ls Used to Assess Expression Levels of Other Isoforms of CXCL12

[0122]HEK293 cells were transfected with 5 different isoforms of CXCL12 (alpha and beta) using commercially available plasmids for each isoform (plasmids available from GenScript). Transfected cells were selected with 250 ug / mL of G418 (commercially available from ThermoFisher) and a stable pool for each isoform was created. Cells were allowed to condition in a suitable medium for 3 days. The conditioned medium was separated in a 4-8% NuPage gel (commercially available from ThermoFisher) with MES buffer and transferred to nitrocellulose (iBLOT).

[0123]Expression levels were detected with HRP labeled, anti-FLAG tag antibody / TMB chromogen (available from GenScript) on a Western Blot, as shown in FIG. 1. The results evidenced that the gamma, delta and theta isoforms of CXCL12 had greater concentrations than the alpha or beta isoforms.

example 3

on of Transgenic Beta Cells

[0124]Pancreatic beta cells derived from human induced pluripotent stem cells were purchased from Takara Bio USA, Inc. (Mountain View, Calif.) and cultured according to provided instructions.

[0125]Cells were transduced with lentiviral vectors (pLenti-C-Myc-DDK, OriGene Technologies, Rockville, Md.) containing a human CXCL12 isotype (CXCL12a / SDF-lalpha or CXCL12b / SDF-lbeta) or control. The lentiviral vectors were used at a ratio of about 10:1 per beta cell. The sequences, including the tag (underlined) are provided below. Concentration of the CXCL12 isotype was determined by ELISA (RayBioTech, Norcross, Ga.) (Table 1).

CXCL12a (aka SDF1a)Accession No. NM_199168SEQ ID NO.: 9ATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCTTTAAACAAGACGCGTACGCGGCCGCTCGAGCA...

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Abstract

Described herein are human transgenic beta cells expressing fugetactic levels of CXCL12 to a subject in need thereof. Also described herein are beta cells comprising a transgene comprising a nucleic acid sequence encoding CXCL12.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application Nos. 62 / 567,604, filed Oct. 3, 2017; 62 / 568,117, filed Oct. 4, 2017; 62 / 637,913, filed Mar. 2, 2018; 62 / 662,651, filed Apr. 25, 2018; 62 / 694,634, filed Jul. 6, 2018; 62 / 696,603, filed Jul. 11, 2018; 62 / 717,587, filed Aug. 10, 2018; 62 / 719,975, filed Aug. 20, 2018; and 62 / 734,910, filed Sep. 21, 2018; each of which is incorporated by reference herein in its entirety.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 25, 2018, is named 054610-501001US SL.txt and is 8,646 bytes in size.FIELD OF THE INVENTION[0003]The invention is directed to genetically modified, human beta cells as well as methods using such cells. The genetically modified (transgenic), human beta cells express a fugetactic amount of a fugetact...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61P3/10C12N5/0781C12N5/0783A61K35/17
CPCC12N5/0676A61P3/10C12N5/0635C12N5/0638C12N5/0646A61K35/17A61K35/39C07K14/522C12N5/0686C12N2502/11C12N2501/998C12N2510/00C12N2502/99
Inventor SWISS, GERALD F.KIEWLICH, DAVID
Owner SDF BIOPHARMA INC
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