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Method

a technology applied in the field of haematopoietic stem cells and haematopoietic progenitor cells, can solve the problems of affecting the function of hsc, limiting allogeneic hct, and difficulties in using the methods employed for hscs genetic modification, etc., to increase gene transfer efficiency, increase transgen

Inactive Publication Date: 2020-10-29
OSPEDALE SAN RAFFAELE SRL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

The patent text describes the discovery that prostaglandin E2 (PGE2) and its derivatives can improve the efficiency of gene transfer into certain types of stem cells. This means that PGE2 and its derivatives can be used to enhance the delivery of genes into cells that can produce blood cells. This discovery could have important uses in research and medicine.

Problems solved by technology

However, allogeneic HCT is limited by the poor availability of matched donors, and the mortality associated with the allogeneic procedure which is mostly related to graft-versus-host disease (GvHD) and infectious complications provoked by the profound and long-lasting state of immune dysfunction.
However, difficulties remain with the methods employed for the genetic modification of HSCs.
However, ex vivo culture negatively impacts on HSC function and this negative effect clearly correlates with the duration of culture (Guenechea G et al.
Ann. N. Y. Acad. Sci. 2012;1266:138-150), the resulting protocols present several challenges for clinical translation, give variable and often poorly reproducible results, and still need to be proven in relevant clinical settings.

Method used

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example 1

Highly Purified HSCs are More Transducible by Lentiviral Vectors

[0312]We studied the differential effect of a microRNA on haematopoietic stem and progenitor cell populations. To this end, CD34+CD38− and CD34+CD38+ cord blood HSPCs were transduced with lentiviral miRNA sponge or overexpressing vectors (data not shown). Unexpectedly, and in contrast to what is widely assumed in the field, we noted a 1.5-fold increased gene transfer into the more primitive, CD34+CD38− HSC-enriched subset. We independently confirmed this observation on multiple cord blood and adult bone marrow donors using biologically neutral vectors expressing marker genes, demonstrating a 1.5 to 2-fold increased gene transfer efficiency into sorted CD34+CD38− HSC-enriched fractions as compared to bulk CD34+ or CD34+CD38+ cell transduction (FIG. 1A).

[0313]Since bulk CD34+ HSPCs contain a small subset of CD38− cells, we wanted to test whether this increased transducibility of more primitive cells was also maintained wi...

example 2

[0316]A bead-based, sequential negative / positive selection for CD38 and CD34, respectively, allows purification of cells with superior NSG engraftment potential

[0317]In order to test the feasibility of a bead-based, sequential negative / positive selection for CD38 and CD34, respectively, we applied a commercially available CD38 selection kit to human cord blood mononuclear cells and tested the engraftment potential of the CD38−(further enriched for CD34 by positive selection) and CD38+ fraction in NSG mice by competitive transplantation (FIG. 2). Even though we used a first generation, non-optimised selection protocol, we could clearly demonstrate an engraftment advantage for the CD38− fraction. While CD38− cells made up less than 20% of the transplant, 70-80% of long-term engraftment was derived from this fraction, motivating further optimisation of this purification protocol.

[0318]Example 3

Modelling a Split Transplant in NSG Mice

[0319]To model the co-transplantation of genetically-...

example 4

[0340]Prostaglandin E2 Increases Gene Transfer into Human Hematopoietic Stem and Progenitor Cells with NSG Repopulating Potential

[0341]In an effort to exploit the anti-apoptotic properties of prostaglandin E2 (PGE2) (Pelus LM et al. Prostaglandins Other Lipid Mediat. 2011;96:3-9), we treated CD34+ HSPC exposed to stress (freeze / thaw cycles, transduction with toxic vectors, electroporation) with the long acting PGE2 homologue 16,16-dimethyl Prostaglandin E2 (dmPGE2; Cayman Chemical, Cat 14750). Unexpectedly, we found a 1.5 fold increased gene transfer efficiency into CD34+ or CD34+CD38− HSPC from cord blood (FIG. 5A) and adult bone marrow (FIG. 6) after dmPGE2 treatment. This difference in vector copy number (VCN) was maintained over more than 4 months after transplantation of the cells into NSG mice, and engraftment levels were not negatively affected by dmPGE2 treatment.

[0342]We have confirmed that G-CSF-mobilised CD34+ peripheral blood stem cells also undergo more efficient transd...

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Abstract

A method of preparing a therapeutic cell population for clinical use from a starting population of cells comprising haematopoietic stem cells, said method comprising separating a population of cells that substantially do not express CD38 but which express CD34 from the starting population of cells, and transducing the separated cell population with a vector to obtain the therapeutic cell population.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 15 / 031,169, filed Apr. 21, 2016; which is a national stage application under 35 U.S.C. § 371 of International Application No. PCT / IB2014 / 065594, filed Oct. 24, 2014; which claims the benefit of United Kingdom Application No. GB 1318830.5, filed Oct. 24, 2013, as well as the benefit of United Kingdom Application No. GB 1409067.4 filed May 21, 2014; the disclosures of each of which are explicitly incorporated by reference herein in their entireties.FIELD OF THE INVENTION[0002]The present invention relates to haematopoietic stem cells (HSCs) and haematopoietic progenitor cells. More specifically, the present invention relates to improved methods for the genetic modification of HSCs. The present invention also relates to improved methods for the use of genetically modified haematopoietic stem and progenitor cells in gene therapy.BACKGROUND TO THE INVENTION[0003]The haematopoietic...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/28C12N5/0789C12N15/86A61K48/00
CPCC12N2510/00C12N15/86A61K48/0091C12N5/0647C12N7/00C12N2740/15043A61K35/28A61P1/02A61P1/04A61P11/02A61P11/06A61P15/00A61P17/00A61P17/02A61P17/06A61P19/02A61P19/10A61P25/00A61P25/28A61P27/02A61P29/00A61P31/00A61P31/18A61P35/00A61P35/04A61P37/08A61P7/04A61P9/00A61P9/10
Inventor NALDINI, LUIGIGENTNER, BERNHARD RUDOLFZONARI, ERIKABOCCALATTE, FRANCESCO
Owner OSPEDALE SAN RAFFAELE SRL
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