Materials and methods relating to the transfer of nucleic...

Details Materials and methods relating to the transfer of nucleic... Materials and methods relating to the transfer of nucleic...

1998-10-05

2004-04-20

Shukla, Ram R. (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S375000, C435S320100, C435S325000, C435S455000, C435S372000

Reexamination Certificate

active

06723561

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to materials and methods for transferring nucleic acid encoding a polypeptide for treating a disease or disorder into populations of quiescent cells such as haematopoietic stem cells (HSCs), using retroviral packaging cell lines and retroviral particles expressing and displaying a growth factor such as stem cell factor (SCF) on the cell surface or as a fusion with a viral envelope protein. The present invention also relates compositions comprising the retroviral packaging cell lines and retroviral particles, and their use in methods of medical treatment, in vivo and ex vivo.
BACKGROUND TO THE INVENTION
The development of somatic gene therapy as a treatment for single gene inherited diseases and some acquired conditions, such as certain types of cancer, represents one of the most important technical advances in medicine. Blood related disorders such as the X-linked immunodeficiencies, or chronic granulomatous disease (CGD), are amongst the most favourable candidates as model systems for the evolution of this technology. The general feasibility of gene therapy for disorders of this type has been amply demonstrated by the results obtained in the treatment adenosine deaminase dependent severe combined immunodeficiency (ADA-SCID) using peripheral blood T-cells.
However, many problems stand in the way of the realisation of the promise of these techniques. For example, in the experiments described above, the T-cells including the genes required by the patients are not immortal, requiring the therapy to be repeated at regular intervals. Further, attempts to effect a permanent correction, for example by gene transfer into pluripotent haematopoietic stem cells (PHSC), have thus far been unsuccessful.
There are a number reasons for this. Firstly, PHSC are very rare in the bone marrow cell population, and so although work has been done on bone marrow cell culture, it is very difficult to draw conclusions from this work regarding PHSCs. Further, in humans there is a dearth of markers to identify PHSC and, at present, the most reliable marker of immature human bone marrow cells is the CD34 antigen, which marks about 1-2% of total marrow cells. However, probably only about 0.1% of these CD34+ cells are true PHSC. In addition, there are no wholly reliable assays for human PHSC, unlike murine systems, where the rescue of lethally irradiated individuals can be used to test for PHSC.
Recently, a method to enrich for PHSC has been described by Beradi et al (Science, 267, 104-108, (1995)) which exploits the quiescence of PHSCs as a basis for their functional isolation. In this method, bone marrow cells were incubated for 7 days in the presence of the cytokines stem cell factor (SCF) and IL-3, to stimulate division in all of the progenitor cells, but not in true PHSC. The cytotoxic agent, 5-fluorouracil (5-FU), was then added to these cultures, resulting in the death of all dividing cells in the culture. However, quiescent cells, including PHSC which average only 1 in 10
5
of the original cells, were spared in this process. Accordingly, the authors reported obtaining an enriched population of cells having the characteristics of true PHSC.
However, the authors of this paper were unable to find any combination of cytokines that was able to stimulate these cells to divide, other than incubation in long term marrow culture (LTC), which also leads to their differentiation.
Thus, although, this method produces highly enriched populations of PHSC, it is their quiescence, the very property exploited for their isolation by Beradi et al, that still represents the most significant hurdle limiting current gene therapy protocols. This is because most highly developed vector systems presently used for gene transduction are based on murine retroviruses and these viruses (and the vectors derived from them) are unable to stably integrate their genome into non-dividing cells, rendering PHSCs refractory to retroviral gene transfer.
Previously, we presented an abstract at the European Working Group for Gene Therapy in November 1994 disclosing that a retroviral cell line containing a viral vector incorporating nucleic acid encoding GCD and expressing stem cell factor on its surface was able to achieve improved rates of transduction in a bone marrow cell culture. However, as mentioned above this cell culture contains a very low proportion of PHSC, and this treatment would not be expected to stimulate the PHSC to divide or to allow the stable integration of the nucleic acid encoding GCD into the PHSC genome. An important fact underlying this expectation is that in Beradi et al, stem cell factor was one of the cytokines used to stimulate selectively division in the most of the cells in marrow cell culture (but not the PHSC), allowing them to be killed to leave the enriched population of stem cells.
SUMMARY OF THE INVENTION
The present invention is based on the unexpected finding that it is possible to get haematopoeitic stem cells to cycle transiently during the period of exposure to vectors incorporating nucleic acid encoding a desired protein or polypeptide by exposing them to bound growth factors such as stem cell factor. This observation means that contrary to prior expectations, a population of quiescent cells such as PHSC can be used as targets for vectors incorporating nucleic acid encoding a desired protein or polypeptide, provided that the cells are additionally exposed to a surface bound growth factor, e.g. stem cell factor expressed by a retroviral packaging cell line so that it is bound on the cell surface or expressed as a fusion with an envelope protein of retrovirus so that the growth factor is displayed on the surface of the retrovirus.
Without wishing to be bound by any particular theory, we believe that the exposure of the quiescent cells to the membrane or surface bound growth factor induces them to start dividing, so that the nucleic acid, e.g. packaged in retroviral particles produced by a retroviral packaging cell line, can infect the cells and become incorporated into their genomes which become accessible during cellular division when the nuclear membrane dissolves. This method has the advantage that it can be adapted for the treatment of a wide variety of disorders, by incorporating nucleic acid encoding an appropriate protein or polypeptide into the vector. A further advantage of the method is that by stimulating the quiescent cells to differentiate at the time of gene transfer, preferential amplification of the transduced cells relative to the non-transduced cells can be achieved.
Accordingly, in a first aspect, the present invention provides a retroviral packaging cell line transformed with a viral vector comprising nucleic acid encoding a polypeptide for treating a disease or disorder, the retroviral packaging cell line being capable of expressing nucleic acid encoding a growth factor so that the growth factor is (i) displayed on the cell surface or (ii) expressed as a fusion with a viral envelope protein so that the growth factor is displayed on the surface of viral particles,
wherein the cell line packages the nucleic acid encoding the polypeptide in viral particles produced by the retroviral packaging cell line, the cell line being for use in a method of medical treatment of a disease or disorder that responds to the polypeptide.
In this aspect, the retroviral packaging cell line includes nucleic acid encoding viral envelope protein so that the cell line can produce viral particles and package the nucleic acid encoding the polypeptide for treating the disease or disorder in them.
In this application, “quiescent” refers to cells that are unlikely to enter mitosis within the next 24 hours in the absence of appropriate growth stimulus. Preferably, the population of quiescent cells are enriched in haematopoeitic stem cells, for instance by employing the isolation method of Beradi et al (supra) using bone marrow cells. Other quiescent cell types suitable for use in the invention include resting T-lymphocytes, B-lymphocytes and monocytes,

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