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Title:  Use of marrow-derived glial progenitor cells as gene delivery vehicles into the central nervous system
United States Patent: 
7,022,321
Issued: 
April 4, 2006
Inventors:
 Eglitis; Martin A. (8573 Twin Pointe Cir., Indianapolis, IN 46236); Mezey; Eva (6515 Old Farm La., Rockville, MD 20852); Mouradian; Mary Maral (9304 Wildoak Dr., Bethesda, MD 20814)
Appl. No.: 
122703
Filed: 
April 11, 2002


 

George Washington University's Healthcare MBA


Abstract

The present disclosure relates to a method for introducing a hematopoietic cell into the brain of a mammal, by administering bone marrow-derived progenitor cells into the body of the mammal by intravenous injection. The bone marrow-derived cell is preferably a cell that differentiates into a glial cell. The disclosure also relates to a method for delivery of therapeutic protein molecules into the brain of a mammal, by administering to a mammal an effective amount of bone marrow-derived progenitor cells which contain a gene having a nucleic acid sequence that encodes a functional therapeutic protein. Isolated recombinant cells and a pharmaceutical composition are also provided.

Description of the Invention

FIELD OF THE INVENTION

The present disclosure relates to methods for introducing hematopoietic cells into the brain of a mammal, the differentiation of adult bone marrow cells into glial cells, and the use of marrow-derived glial progenitor cells as gene delivery vehicles into the brain.

BACKGROUND OF THE INVENTION

Glial cells are thought to derive embryologically from either myeloid cells of the hematopoietic system (microglia) or neuroepithelial progenitor cells (astroglia and oligodendrocytes). However, it is unclear whether the glia in adult brains free of disease or injury originate solely from cells present in the brain since the fetal stage of development, or if there is further input into such adult brains from cells originating outside the central nervous system (CNS).

Besides the cells of the vasculature, the brain comprises two general cell types: neurons and glial cells. Glial cells provide physiological support to neurons and repair neuronal damage due to injury or disease. Macroglia (astroglia and oligodendroglia) are generally considered to be derived from neuro-ectoderm and are believed to be developmentally distinct from microglia (1). However, the developmental origin of microglia remains debatable (2,3). The two major views are that they derive either from neuro-epithelial cells (4-6) or from hematopoietic cells (i.e., monocytes) (7,8). The extent to which cells outside the CNS contribute to the maintenance of microglia in adults remains debatable (compare (9) and (10)), and no such contribution to adult neurons or macroglia has been previously described.

SUMMARY OF THE DISCLOSURE

Heretofore, gene therapy in the brain relied upon surgically implanting the transfected cells into the recipient brain. It was unknown prior to our disclosure that cells of the hematopoietic system are a source of progenitor cells for the CNS, such that these cells can be used as a gene therapy delivery vehicle into the brain.

We tested the ability of hematopoietic cells to contribute to the CNS, by transplanting adult female mice with donor bone marrow cells genetically marked either with a retroviral tag or by using male donor cells. We monitored the appearance of the cells in the brain using in situ hybridization histochemistry (ISHH) combined with immunohistochemistry. We also performed double ISHH with digoxigenin and radioactively labeled probes to analyze which cell types might be derived from bone marrow stem cells. We detected a continuing influx of hematopoietic cells into the brain. Marrow-derived cells were already detected in the brains of mice three days after transplant and their numbers increased over the next several weeks, exceeding 14,000 cells per brain in several animals. Marrow-derived cells were widely distributed throughout the brain, including the cortex, hippocampus, thalamus, brainstem, and cerebellum. When ISHH was combined with immunohistochemical staining using lineage-specific markers, some bone marrow-derived cells were positive for the microglial antigenic marker F4/80. Other marrow-derived cells surprisingly expressed the astroglial marker glial fibrillary acidic protein (GFAP). These results indicate that some microglia and astroglia arise from a precursor that is a normal constituent of adult bone marrow.

The results reported here confirm that cells derived from the bone marrow can migrate into the brains of adult mice. Furthermore, we have found that this migration is rapid, with numerous cells present by the third day after transplant. These new cells are distributed throughout the brain, and appear to reside within the parenchyma, since perfusion with PBS does not remove them. Occasional donor marrow-derived cells were found in association with vascular structures. Moreover, densities of donor cells in the parenchyma paralleled the capillary density of a given region. For instance, cortex, with fewer capillaries, had a lower cell density than the more vascularized choroid plexus. Regions with a higher capillary density, such as the area postrema, also had the highest density of marrow-derived cells within the parenchyma.

Double-labeling analyses show that at least some bone marrow-derived cells acquire microglial antigenic markers. However, we also observed many cells positively labeled by ISHH that did not express the F4/80 antigen. This may be due simply to a level of antigen below the limits of detection in our assay.

Alternatively, it is possible that the F4/80 marker is expressed on marrow-derived cells only after they fully differentiate into microglia, while less mature microglial precursors are not recognized by the antibody to F4/80. Nonetheless, our results strongly support the view that hematopoietic cells outside the CNS contribute to the maintenance of microglia in healthy adults. While a partial CNS origin of adult microglia cannot be excluded, our data is inconsistent with an exclusively CNS origin. Moreover, although our experiments did not examine fetal origins of microglia, the finding of hematopoietically-derived microglia in healthy adults is also consistent with a hematopoietic origin of microglia in development.

Surprisingly, we found that some hematopoietic cells (tagged either with a retroviral vector or by transplant of male cells into a female recipient) give rise to cells other than microglia, specifically to cells that exhibit astroglial markers. Although this observation is unexpected, it is based on identical results in multiple animals using two independent means of cell tagging with both cytoplasmic and nuclear markers.

The appearance of marrow-derived astroglia seems a normal process in these animals. Because the number of marrow-derived cells detected in the brain increased over time, their appearance does not appear to be a consequence of the transplantation procedure itself. If appearance in the brain was a by-product of transplantation, one would expect tagged cell numbers in the brain to peak and then decline, which was not observed. Rather, the data is consistent with existence of cells, amongst the populations of marrow- engrafting cells, capable of continuous generation of progenitors that migrated to the brain. Interestingly, cells with marrow markers were seen in the ventricular ependyma. In fact, in many animals, marrow-derived cells could be found concentrated sub-ependymally (Mezey & Eglitis, in preparation). The subependymal zone is an important source of neuronal and glial progenitors during development (24) and in adults (27). Finding bone-marrow derived cells in this location opens the possibility that such cells receive cues guiding their differentiation once they enter the brain. Studies evaluating this possibility are ongoing.

No obvious pathology such as gliosis was detected in the brain of any transplant recipient (n=46). Some recipient animals were irradiated before receiving bone marrow transplants to see if marrow purging enhanced engraftment and seeding of implanted cells. However, radiation dosages were at least one order of magnitude below those known to induce pathological changes in the CNS (29). Indeed, we found preconditioning of recipients was not necessary. Male donor cells engrafted and persisted for at least 10 weeks even without irradiation. Furthermore, as many Y chromosome/GFAP double positive cells were seen with as without irradiation. The wide distribution of GFAP-positive cells in both gray and white matter demonstrates that bone marrow-derived progenitors are not restricted to differentiate into a particular subclass of astroglia. That is, marrow-marked cells contributed to both fibrous astrocytes in the white matter and protoplasmic astrocytes in the gray matter.

One alternative explanation for our observing GFAP staining of cells bearing marrow markers is that processes from endogenous astroglia surround the in-migrating cells from the donor marrow. However, some of our data argue against this possibility. First, cytoplasmic neoR ISHH labeling coincided with cytoplasmic GFAP immunostaining. Furthermore, upon evaluation of fifty to 100 male nuclei associated with GFAP staining, no nuclei were seen that could be considered part of an engulfing astroglial cell. If endogenous astroglia were the source of the GFAP staining associated with donor male nuclei, one would expect the geometry in 12μ sections to reveal the cell body and nucleus corresponding to the putative engulfing processes in at least a few cases. After analyzing dozens of sections, no such cases were observed.

Because only about 10% of marrow-derived cells in the brain exhibit expression of either the microglial F4/80 antigen or the astroglial marker GFAP, the identity of the majority of bone marrow-derived cells remains an open question. Nonetheless, there is clearly a measurable contribution by cells of hematopoietic origin to the glial cell population of the brain in adult mice, which indicates that some glial progenitors reside outside the CNS. The observation of marrow-derived astroglia in the optic tract demonstrates that some of these marrow-derived progenitors may be similar to the previously recognized astroglial precursor (30).

Microglia and astroglia respond differently to brain injury. In fact, astrogliosis often appears to be a response to primary microgliosis (31,32). There is also evidence that different brain lesions elicit different microglial and astroglial responses (33). Our results provide a way that gene transfer into hematopoietic progenitors can be used to introduce genes into microglia and astroglia that then would participate in the gliosis associated with a CNS pathology. The detection of marrow-derived cells in brains within days of transplantation provides a method in which genetically altered hematopoietic cells could be used to treat acute diseases of the brain.

Although many neurotrophic factors show promise in the treatment of CNS disorders, their use has been hindered by their inability to cross the blood-brain barrier and by their limited diffusion into CNS tissues (34). In addition, adverse effects have been reported after systemic administration of some neurotrophins (35). Using marrow-derived cells to deliver therapeutic proteins directly to the site of CNS pathology likely would be more benign than systemic administration of toxic molecules. In addition, using vectors with cell type-specific promoters could restrict gene expression specifically to reactive astroglia or microglia, thereby providing greater therapeutic precision for gene therapy of CNS disease.
 


Claim 1 of 8 Claims

1. A method of treating Parkinson's disease, comprising:

transfecting harvested bone marrow cells with a retroviral vector comprising a gene for glial cell line-derived neurotrophic factor (GDNF);

administering the transfected cells intravenously to a subject having Parkinson's disease; and

allowing the transfected cells to migrate to the brain of the subject and express the GDNF gene, thereby treating the Parkinson's disease.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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