The United States of America, as Represented by the Secretary, Department of Health and Human Services

Elixirgen Therapeutics, Inc.

Elixirgen, LLC

Minoru S H Ko

Michal Zalzman

Tomokazu Amano

Lioudmila V Sharova

Tetsuya Hirata

Shigeru B H Ko

Geppino Falco

Sung-Lim Lee

Manuela Monti

Ilaria Stanghellini

The present disclosure relates to methods for transiently activating temperature-sensitive agents in one or more cells, for example by contacting one or more cells with a temperature-sensitive agent and transiently incubating the cells at a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. Additionally, the present disclosure relates to methods of contacting one or more cells in a subject with a temperature-sensitive agent and then lowering the subject's core body temperature to a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. The disclosure also relates to methods of contacting one or more cells in a subject with a temperature-sensitive agent, maintaining the subject's surface body temperature at a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. Further disclosed are methods of treating a subject with a temperature-sensitive therapeutic agent.

Temperature-based transient delivery of nucleic acids and proteins to cells and tissues

The present disclosure relates to methods for increasing telomere length in one or more human cells and/or increasing genome stability of one or more human cells, for example by contacting one or more human cells with an agent that increases expression of Zscan4 in the one or more human cells. Methods of treating a subject in need of telomere lengthening, treating a disease or condition associated with a genomic and/or chromosome abnormality, of rejuvenating one or more human cells, of rejuvenating tissues or organs, and of rejuvenating a subject in need thereof, for example by contacting one or more human cells in the subject with an agent that increases expression of Zscan4, or by administering to a subject in need thereof, an agent that increases expression of Zscan4 are also provided.

Methods of using ZSCAN4 for rejuvenating human cells

Described herein is the finding that increasing the frequency of Zscan4 activation in mouse ES cells not only enhances, but also maintains their developmental potency in long-term cell culture. As the potency increases, even a whole animal can be produced from a single ES cell injected into a 4N blastocyst at an unexpectedly high success rate. The studies disclosed herein indicate that ES cells acquire higher potency by going through the transient Zscan4 activation state more frequently than the regular state. Particularly disclosed herein is the finding that the constitutive presence of Zscan4-ERT2, even in the absence of its usual activator tamoxifen, can increase the frequency of endogenous Zscan4 activation in ES cells, resulting in the increase of developmental potency of the ES cells. Accordingly, provided herein are Zscan4-ERT2 fusion proteins and nucleic acid molecules and vectors encoding Zscan4-ERT2 fusion proteins. Further provided are methods of prolonging and/or enhancing stem cell pluripotency using the disclosed Zscan4-ERT2 nucleic acid molecules and fusion proteins.

Methods for enhancing the pluripotency of stem cells

Described herein is the finding that increasing the frequency of Zscan4 activation in mouse ES cells not only enhances, but also maintains their developmental potency in long-term cell culture. Particularly disclosed herein is the finding that the constitutive presence of Zscan4-ERT2, even in the absence of its usual activator tamoxifen, can increase the frequency of endogenous Zscan4 activation in ES cells, resulting in the increase of developmental potency of the ES cells. Accordingly, provided herein are Zscan4-ERT2 fusion proteins and nucleic acid molecules and vectors encoding Zscan4-ERT2 fusion proteins. Further provided are methods of prolonging and/or enhancing stem cell plmipotency using the disclosed Zscan4-ERT2 nucleic acid molecules and fusion proteins.

Compositions for enhancing the pluripotency of stem cells

The present disclosure relates to methods for increasing telomere length in one or more human adult cells and/or increasing genome stability of one or more human adult cells, for example by contacting one or more human adult cells with an agent that increases expression of Zscan4 in the one or more human adult cells. Methods of treating a subject in need of telomere lengthening, treating a disease or condition associated with a telomere abnormality, of rejuvenating one or more human adult cells, of rejuvenating tissues or organs, and of rejuvenating a subject in need thereof, for example by contacting one or more human adult cells in the subject with an agent that increases expression of Zscan4, or by administering to a subject in need thereof, an agent that increases expression of Zscan4 is also provided.

ZSCAN4, a gene expressed in ES cells and 2-cell stage embryos, has been previously shown to regulate telomere elongation and genome stability in mouse ES cells. It is disclosed herein that in the adult human pancreas, a small number of ZSCAN4-positive cells are present among cells located in the islets of Langerhans, acini, and ducts. These data disclosed herein indicates that expression of ZSCAN4 is a marker for rare stem/progenitor cells in adult human pancreas. Thus, provided herein is a method of isolating pancreatic stem cells or progenitor cell from a sample by detecting expression of ZSCAN4. Also provided is a method of treating diabetes by isolating ZSCAN4pancreatic stem cells or progenitor cells, expanding the cells in vitro and transplanted the expanded cells into the subject. The expanded ZSCAN4cells can optionally be differentiated into pancreatic &#x3b2; cells before transplanting the cells into the subject. Further provided is an in vivo method of expanding a population of pancreatic stem cells or progenitor cells in a subject by delivering a ZSCAN4 protein, a ZSCAN4 nucleic acid sequence, or an agent that increases expression of ZSCAN4 to the pancreas of the subject.

ZSCAN4 as a marker for pancreatic stem cells and progenitor cells and use thereof

Disclosed herein is the finding that Zscan4 is an early embryonic factor that facilitates cellular reprogramming. In particular, Zscan4 can replace the oncogenic reprogramming factor c-Myc to produce induced pluripotent stem cells when co-expressed with Klf4, Oct4 and Sox2. In addition, several Zscan4-dependent genes were identified that promote iPSC formation when co-expressed with known reprogramming factors. Thus, the present disclosure provides an ex vivo method of producing an iPS cell by reprogramming of a somatic cell. The method includes contacting the somatic cell with a Zscan4, or a Zscan4-dependent gene, and at least one reprogramming factor. Also provided are iPS cells produced by the disclosed method and non-human animals generated from such iPS cells.

Use of Zscan4 and Zscan4-dependent genes for direct reprogramming of somatic cells

The disclosure provides methods for increasing genome stability of an embryonic stem (ES) cell or induced pluripotent stem (iPS) cell, increasing telomere length in an ES or iPS cell, or both, for example by contacting an ES or iPS cell with an agent that increases expression of Zscan4 in the cell. Methods for increasing genome stability or increasing telomere length in a population of ES or iPS cells are provided, for example by selecting Zscan4ES or iPS cells from the population of ES or iPS cells (which can include both Zscan4and Zscan4ES or iPS cells). Therapeutic methods of using ES or iPS cells expressing Zscan4 are also provided. Further provided are methods of treating cancer by administering a Zscan4 polynucleotide or Zscan4 polypeptide. Also provided are methods of inducing differentiation of isolated ES or iPS cells into germ cells.

Methods for enhancing genome stability and telomere elongation in embryonic stem cells

The disclosure provides methods for increasing genome stability of an embryonic stem (ES) cell or induced pluripotent stem (iPS) cell, increasing telomere length in an ES or iPS cell, or both, for example by contacting an ES or iPS cell with an agent that increases expression of Zscan4 in the cell. Methods for increasing the genome stability in a population of ES or iPS cells, increasing telomere length in a population of ES or iPS cells, or both, are provided, for example by selecting Zscan4ES or iPS cells from the population of ES or iPS cells (which can include both Zscan4and Zscan4ES or iPS cells). Therapeutic methods of using ES or iPS cells expressing Zscan4 are also provided. Further provided are methods of treating cancer by administering a Zscan4 polynucleotide or Zscan4 polypeptide. Also provided are methods of inducing differentiation of isolated ES or iPS cells into germ cells.

Described herein is Zscan4, a gene exhibiting 2-cell embryonic stage and embryonic stem cell specific expression. Identification of nine Zscan4 co-expressed genes is also described. Inhibition of Zscan4 expression inhibits the 2-cell to 4-cell embryonic transition and prevents blastocyst implantation, expansion and outgrowth. Provided herein are methods of inhibiting differentiation of a stem cell, promoting blastocyst outgrowth of embryonic stem cells and identifying a subpopulation of stem cells expressing Zscan4. Further described is the identification of Trim43 as a gene exhibiting morula-specific expression. Also provided are isolated expression vectors comprising a Zscan4 promoter, or a Trim43 promoter operably linked to a heterologous polypeptide and uses thereof. Further provided are transgenic animals comprising transgenes encoding marker proteins operably linked to Zscan4 and Trim43 promoters.

Growing community of inventors

Cockeysville, MD, United States of America

Minoru S H Ko