Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-08-31
2003-09-30
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S325000, C435S372000, C435S320100
Reexamination Certificate
active
06627442
ABSTRACT:
TECHNICAL FIELD
The present invention is directed to methods, as well as compositions related thereto, for the efficient and stable transduction of cells using viral vectors. The methods increase the efficiency of transduction by contacting the cell to be transduced with one or more molecules that bind the cell surface. The contacting step may occur before, after, or simultaneously with, introduction of the viral vector to the cells. The present invention also concerns the use of the stably transduced cells in other applications, including expression of nucleic acids borne by the vector or therapy of living organisms.
BACKGROUND ART
“Transfection”, which generally refers to techniques for the introduction of genetic material into a cell, has contributed greatly to the molecular and recombinant revolutions in biology. Examples of transfection techniques for use with higher eukaryotic cells include calcium phosphate precipitation, DEAE-dextran treatment, electroporation, microinjection, lipofection, viral infection, and other methods found in numerous scientific textbooks and journals.
Among transfection techniques, the use of viral infection is unique in that a virus naturally occurring means of introducing its genetic material into a cell is taken advantage of to transfer a nucleic acid molecule of interest into a cell. Examples of viruses modified and applied to such techniques include adenoviruses, adeno-associated viruses, herpes simplex viruses, and retroviruses. Generally, nucleic acid molecules of interest may be cloned into a viral genome. Upon replication and packaging of the viral genome, the resultant viral particle is capable of delivering the nucleic acid of interest into a cell via the viral entry mechanism.
Commonly, the viral genome is first made replication deficient by nucleic acid manipulation before the addition of the nucleic acid of interest. The resultant viral genome, or viral vector, requires the use of a helper virus or a packaging system to complete viral particle assembly and release from a cell. When a viral vector or viral particle is used to transfer genetic material of interest into a cell, the technique is referred to as “transduction”. Thus generally, to “transduce” a cell is to use a viral vector or viral particle to transfer genetic material into a cell.
Among transduction techniques, the use of retroviruses has been the subject of great interest for the genetic modification of mammalian cells. Of particular interest is the use of modified retroviruses to introduce genetic material into cells to treat genetic defects and other diseases. An example of this approach is seen in the case of cells of the hematopoietic system, where retroviruses and lentiviral vectors are the subject of intense research.
Movassagh et al., for example, discuss their studies on their attempts to increase the efficiency of retrovirus mediated transduction by including results from studies on the cell cycle of activated T cells. As such, their results are dependent upon active cell division during transduction. The work is also limited to the use of a murine onco-retrovirus and the requirement for significant prestimulation of the cells before transduction.
June et al. (WO 96/34970) describe the use of T cell stimulation as a means to increase T cell transfection. Other work on T cell transduction with activated or stimulated cells include those of Douglas et al., Hooijberg et al, Onodera et al., Klebba et al., Barry et al., and Unutmaz et al. Unfortunately, none of this work demonstrated transduction efficiencies of greater than about 65%.
Costello et al. describe the transduction of both stimulated and non-stimulated T cells using Human Immunodeficiency Virus-1 (HIV-1) lentiviral vectors. They observed only about a maximum of 17% efficiency with stimulated primary T cells and less than 19% efficiency with non-stimulated T cells. They also noted a limited ability to increase efficiency to no more than 36% in stimulated T cells by including the presence of HIV-1 accessory proteins.
Chinnasamy et al. also describe an increase in the efficiency of transduction in the presence of HIV-1 accessory proteins in both non-stimulated and mitogen stimulated T cells. Like Movassagh et al, Chinnasamy et al. prestimulated blood lymphocytes for significant periods prior to transduction with a lentiviral vector. While Chinnasamy et al. initially observed a greater than 96% transduction efficiency three days after transduction, the percentage of stably transduced cells decreased to 71.2% two weeks after transduction. Haas et al. also observed transient transduction and “pseudotransduction” in cells transduced with a lentiviral vector capable of expressing a marker gene (green fluorescent protein). Even three days post transduction, significant (over 10%) transient transduction was detected based on non-integrative expression of the marker gene in transduced primary CD34+ cord blood cells. Such expression from transient transduction remained detectable at about 5% even seven days post-transduction. Only after about 10 days post transduction did expression from transient transduction mirror that in cells transduced with a markerless vector.
Therefore, Chinnasamy et al were not able to achieve stable transduction, where an integrated form of the viral vector has been inserted into the chromosomal DNA of the transduced cell, of primary lymphocytes beyond 71.2% as reflected by the efficiency after two weeks. This was despite the use of cytokines to prestimulated the cells. Furthermore, Chinnasamy describe their inability to significantly transduce (only 3.6% 14 days post transduction) non-stimulated lymphocytes with a HIV vector that did not express accessory proteins (Vif, Vpr, Vpu and Nef), even though the cells were later stimulated with the PHA mitogen and the IL-2 cytokine post-transduction. While the results were improved somewhat with the use of non-stimulated cells and vectors containing accessory proteins, in no case was the efficiency of stable transduction of stimulated or non-stimulated cells greater than 75% on day 14 post transduction, irrespective of the stimulatory protocol used with the vector.
Low frequencies of stable transduction with lentiviral vectors was also observed by Hass et al., who could only achieve a maximum stable transduction efficiency of less than 25%, seven days post transduction, with primary CD34 positive cord blood cells. Strikingly, this 25% upper limit of transduction could not be improved even after extremely high multiplicities of infection or vector concentrations, such as a multiplicity of infection (MOI) of up to 9000 and vector concentrations of up to 10
8
infectious units per milliliter.
Follenzi et al. also used a very high MOI of 500 to transduce cells in the presence of a three cytokine cocktail containing interleukin-3 (IL-3), interleukin-6 (-IL6) and stem cell factor (SCF). Interestingly, use of the cocktail would render the cells unsuitable for human clinical transplantation.
Thus there remains a need to provide a more efficient means of stably transducing cells with vectors at high frequency. Additionally, there is a need for a more efficient means to transduce non-stimulated cells for use both as research tools and as a therapeutic agent.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
DISCLOSURE OF THE INVENTION
The present invention provides highly efficient methods, and compositions related thereto, for the stable transduction of cells with viral vectors and viral particles. By “stable transduction,” it is meant where an integrated form of the viral vector has been inserted into the chromosomal DNA of the transduced cell. The methods comprise exposing the cells to be transduced to contact with at least one molecule that
Chang Yung-Nien
Davis Brian
Dropulic Boro
Han Wei
Humeau Laurent
LeGuyader John L.
Morrison & Foerster / LLP
Schmidt Mary
VIRxSYS Corporation
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