Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2002-02-12
2004-07-27
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S366000, C435S368000, C435S378000
Reexamination Certificate
active
06767738
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the fields of stem cells and gene therapy.
BACKGROUND OF THE INVENTION
Cell proliferation in the adult mammalian brain is ubiquitous but is largely confined to the measured production of glia. Except for discrete regions in the hippocampus and the subventricular zone (SVZ), neurogenesis is conspicuously absent. The reasons why these areas continue to generate neurons are unknown, but primary cell cultures from the adult rodent brain are beginning to provide some insights. Cultures initiated from adult SVZ or hippocampal tissues contain proliferative neuronal and glial-restricted progenitors as well as multipotent precursors with the characteristics of neural stem cells, i.e., the ability to self-renew and the ability to generate both neurons and glia.
It has been suggested that stem cells may be more widely distributed since cells from non-neurogenic areas repeatedly passaged in the presence of high concentrations of basic fibroblast growth factor (FGF-2) appear to begin to generate neurons in vitro. This observation is consistent with the isolation of neuronal progenitors from these areas, but the protracted times in culture suggests another explanation. It is known that stem cell cultures initiated from hippocampal tissues will spontaneously transform, due to accumulated genetic abnormalities. Abnormalities in chromosome number can occur in as little as 30 population doublings and, as cells become increasingly aneuploid, it is possible that glial-restricted progenitors acquire capabilities beyond those available in vivo.
With existing methodologies, it has been difficult to distinguish between the activation of a latent potential vs. in vitro mutation. Unlike fibroblast tissues, which are easily dissociated and yield relatively abundant precursor populations, adult tissues yield few progenitors and the progenitor preparations are contaminated with differentiated cells and tissue debris. The myelin-rich debris inhibits cell attachment and growth while differentiated cells complicate the evaluation of lineage potential in acutely isolated cultures. Past studies have evaluated “progenitors” only after repeated passaging had eliminated the debris and differentiated cells. Even if these cells had remained diploid, they may have been dramatically altered in prolonged culture. Accordingly, there exists a need in the art for isolating a “clean” (i.e., more enriched) cell population containing putative progenitor and stem cells, so that they can be studied without contaminating debris.
The ability to isolate progenitor or stem cells from a variety of tissues would provide a basis for therapeutic applications using these immature cell types. Neural tissue, in particular, comprises a unique biological system that presents unique therapeutic challenges. Damaged neural tissue has proven very difficult to repair or replace. To facilitate the repair or replacement of neural tissue, scientists have focused their efforts on the identification, isolation and use of neuronal stem cells (or “progenitor cells”). With an appropriately pluripotent neural progenitor or stem cell, regeneration or augmentation of a variety of neural cell types is a possibility. Indeed, with a progenitor cell exhibiting an even more widely ranging plasticity, the regeneration or augmentation of a variety of cell types should be possible. Similarly, the stem cell would be a useful vehicle for introducing exogenous genetic material, as desired to achieve therapeutic results.
Occular tissue, in particular the retina, represents a highly specialized neural structure for which repair is often required. For example, the eye is frequently subjected to environmentally or genetically induced injury. As a result, appropriately plastic stem cells would present a valuable vehicle for repair, replacement and/or genetic manipulation (e.g., gene therapy). Although physical damage to the eye may require merely replacing damaged cells with a cell type exhibiting the required plasticity, genetically mediated degeneration of occular tissue presents a more complex challenge.
In many instances, the exact molecular mechanisms that mediate occular or retinal degeneration are poorly understood. Grafting genetically modified cells provides an effective method for evaluating the relative impact of candidate molecules on retinal biology. In addition, the ability to introduce engineered cells may provide a considerable therapeutic benefit for a variety of progressive degenerative diseases. Past attempts to use cell grafts for the delivery of transgene products in the eye have met with mixed success. Heterotypic grafts of non-neural cells fail to integrate and often physically disrupt the normal retinal architecture; Planck, S. R., et al.
Curr. Eye Res
. 11: 1031 (1992). Homotypic fibroblast tissue grafts show some integration but are not amenable to genetic manipulation prior to implantation. See, for example, Seiler, M. J. & Aramant, R. B., Transplantation of embryonic retinal donor cells labelled with BrdU or carrying a genetic marker to adult retina, Exp. Brain Res. 105: 59-66 (1995); Gouras, P., Du, J., Kjeldbye, H., Yamamoto, S., Zack, D. J., Long-term photoreceptor transplants in dystrophic and normal mouse retina, 35:3145-3153 (1994); and Gouras, P., Du, J., Kjeldbye, H., Kwun R., Lopez, R., Zack, D. J., Transplanted photoreceptors identified in dystrophic mouse retina by a transgenic reporter gene,
Invest Ophthalmol. Vis. Sci
. 32: 3167-3174 (1991). Grafts of retinal cell lines (i.e., immortalized cell lines) partially overcome these problems. Trisler, D., Rutin, J. & Pessac, B., Retinal engineering: engrafted neural cell lines locate in appropriate layers,
Proc. Natl. Acad. Sci. U.S.A
. 93: 6269-274 (1996); del Cerro, M., Notter, M. F., Seigel, G., Lazar, E., Chader, G., del Cerro, C., Intraretinal xenografts of differentiated human retinoblastoma cells integrate with the host retina,
Brain Res
., 583:12-22 (1992). However, immortalized cells present potential risks of tumor formation that makes their use less than ideal.
Several groups have reported heterotypic transplants of neuronal progenitor cells, immortalized neural cell lines or embryonic neural precursors into the CNS. To date, however, none of these studies has shown satisfactory diversity and distribution of cell types to render such cells broadly useful either as cell source for cell replacement therapy or-for the expression of transgenes in the host tissue.
In recent years, significant attention has been directed toward identification and characterization of very immature progenitor cells in the adult brain. Gage, F. H., Ray, J. & Fisher, L. J., Isolation, characterization, and use of stem cells from the CNS,
Annu. Rev. Neurosci
., 18:159-192 (1995). Additionally, it has been reported that stem cell-like multipotent progenitors can be isolated from adult hippocampus of rats, expanded in vitro and subsequently grafted into adult hippocampus and olfactory bulb where they demonstrate site-specific neuronal differentiation. See, for example, Palmer, T. D., Talkahashi, J., Gage, F. H., The rat hippocampus contains primordial neural stem cells,
Mol. Cell. Neurosci
., 8: 389 (1997); Palmer, T. D., Ray, J. & Gage, F. H. FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain,
Mol. Cell Neurosci
. 6: 474-486 (1995); Gage, F. H., Coates, P. W., Palmer, T. D., et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.
Proc. Natl. Acad. Sci. U. S. A.
92:11879-11883 (1995); and Suhonen, J. O., Peterson, D. A., Ray,J. & Gage, F. H. Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo, Nature 383:624-627 (1996). Some of the phenotypes generated in the olfactory bulb are not found in the hippocampus, suggesting that the most immature of these stem-like cells may retain considerable plasticity.
As part of the central nervous system, both developmentally and phenotypically, the retina shares the
Gage Fred H.
Palmer Theo
Safar Francis G.
Takahashi Jun
Takahashi Masayo
Kunz Gary
Nichols Christopher James
Reiter Stephen E.
The Salk Institute for Biological Studies
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