Homologous recombination in multipotent adult progenitor cells

Abstract

The invention relates to methods of altering gene expression by homologous recombination in a multipotent adult progenitor cell (MAPC). In particular, methods of producing a recombinant MAPC, of correcting a genetic defect in a mammal, of providing a functional and / or therapeutic protein to a mammal, and of transforming a MAPC are provided. MAPCs containing an erogenous DNA as well as recombinant MAPCs and their differentiated progeny are also provided.

Description

RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. application Ser. No. 60 / 429,631, filed on Nov. 27, 2002. This application makes reference to International Application No. PCT / US00 / 21387, filed Aug. 4, 2000 and to International Application No. PCT / US02 / 04652, filed Feb. 14, 2002, both of which are hereby incorporated by reference. Each document cited or referenced in each of the foregoing applications, and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the foregoing applications and in any of the cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text or in any document incorporated into this text, are incorporated herein by reference. Documents incorporated by reference into this tex...

AI Technical Summary

Benefits of technology

[0019] The ability to repair, alter, replace, delete or express desired nucleotide sequences in the genome of MAPCs would expand the potential usefulness of MAPCs in the in vitro, ex vivo, or in vivo expression of a gene of interest in order to arrive at a multitude of desired outcomes, such as nutritional and / or therapeutic protein production, non-protein gene expression (e.g. generation, up / down regulation, or knock-out / -in of ribozymes), as well as treatment of disease. Use of gene targeting in MAPCs can provide methods for altering gene expression (i.e., by increasing or decreasing the production of a gene product) not only to produce a novel or enhanced gene product, but also for investigation of gene expression patterns and / or gene function.

Problems solved by technology

Stem cells which differentiate only to form cells of the hematopoietic lineage, however, are unable to provide a source of cells for repair of other damaged tissues, for example, heart or lung tissue damaged by high-dose chemotherapeutic agents.

They are also limited in their use in cell-based therapy to the correction of defects that affect only cells of the hematopoietic lineage.

Similarly, their use in in vitro and / or ex vivo protein production is limited to proteins normally expressed in cells of hematopoietic lineage.

These approaches have limitations, such as the potential of generating replication-competent virus during vector production; recombination between the therapeutic virus and endogenous retroviral genomes, potentially generating infectious agents with novel cell specificities, host ranges, or increased virulence and cytotoxicity; limited cloning capacity in the retrovirus (which, inter alia, restricts therapeutic applicability) and short-lived in vivo expression of the product of interest.

Further, in most gene delivery systems, it is not possible to direct or target the donor DNA (i.e., the DNA being delivered to the cell, such as therapeutic DNA) to a preselected site in the genome.

This mixture of infected cells is problematic in two senses: first, since integration site plays a role in the function of the donor DNA, each cell has a different level of function and, second, since the integration of donor DNA into the genome can trigger undesired events, such as the generation of tumorigenic cells, the likelihood of such events is dramatically increased when millions to billions of independent integrations occur.

While this is theoretically possible, success rates for creating a clonal population from a single cell can be low and the number of passages required to amass a usable number of transfected cells can be deleterious.

From a practical standpoint, and due to ethical concerns, germ cell gene targeting is inappropriate for human use.

ES cell gene targeting is also controversial, and the availability of ES cells for these purposes is severely limited.

However, the practical use of somatic cells is limited to conditions that affect only one cell type.

Therefore, for example, an altered somatic cell cannot be induced to differentiate into cells of various tissue types.

In addition, somatic cells are generally limited in their potential to propagate in vitro.

Historically, transfer (or introduction) of exogenous DNA into stem cells has been challenging, with most of the known transfection methods giving sub-optimal transfer rates.

Achieving high rates of transfer in stem cells has been hindered by the fact that optimal transfection occurs when cells are cultured at high density, while lower cell densities are required to maintain stem cells in an undifferentiated state.

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