Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-01-25
2002-12-03
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S069700, C530S387300, C536S023400, C536S023720
Reexamination Certificate
active
06489142
ABSTRACT:
The present invention relates to the field of biotechnology. More particularly, it concerns methods and constructs for producing retroviral particles in vitro, ex vivo or in vivo. It also concerns the use of such methods and constructs for transferring nucleic acids into cells.
Retroviruses are currently one of the main types of vector for transferring nucleic acids into cells. They are used for example for transferring nucleic acids in vitro or ex vivo (experimental studies, study of regulation, production of recombinant proteins, introduction of resistance genes or toxicity genes) or directly in vivo (establishment of animal pathological models, labeling or bioavaiiability studies, therapeutic use by grafting producer cells or injecting purified virus, etc.).
For all these utilizations, it is therefore important to have effective methods available for producing and administering retroviruses. In this respect, conventional methods are based on the use of packaging cell lines. Such cell lines are constructed in vitro and express the whole set of proteins required for constituting and packaging a defective retroviral vector in a retroviral particle. Such cell lines are exemplified by the cell lines PSICRIP [Danos and Mulligan, PNAS (1988) 85: 6460], PA317 [Miller and Buttimore, Mol. Cell. Biol. (1986) 6: 2895], or GP+ Env AM12 [Markowitz et al., Virology (1988) 167: 400]. However, the use of such cell lines can pose certain problems, related first to their construction and second, to their use.
For example, such cell lines must be stable, that is, express the retroviral functions in a continuous manner, without genetic rearrangements or loss of expression. In addition, such cell lines must be compatible with the potential pharmacological use of the retroviruses, and must therefore be established from cells that can be cultured, having little or no immunogenicity, etc.
Furthermore, it is important that the cell lines used produce fairly high viral titers free of replication-competent virus (RCV). The methods of retroviral preparation by means of packaging cell lines therefore necessarily comprise quality controls on both the viral stocks produced and the cell lines used. Finally, depending on the desired application, these production methods may have to be carried out under a high level of confinement, further complicating the industrial scalability of such methods.
To address these drawbacks, patent application WO 95/22617 proposed a new concept for producing retroviral particles that avoids the use of retrovirus packaging cells. This new concept consists in transforming a target cell in vitro, ex vivo or directly in vivo to a retrovirus producing cell, by introducing into this cell the whole set of genetic elements required for constituting a retroviral particle. This application provides in particular for transferring these elements by means of plasmid constructs or viral vectors of different origin. As a matter of fact, Feng et al. [Nature Biotechnology (1997) 15: 866] have described a specific embodiment of this new approach, using two first generation recombinant adenoviruses (i.e. defective for the E1 region) into which the retroviral genetic elements have been distributed in a precise arrangement.
The present application now describes a further improvement to the retroviral particle production methods described hereinabove, and the use for transferring nucleic acids.
The present application is based in particular on the use of recombinant adenoviruses for delivering to cells, in vitro, ex vivo or in vivo, the whole set of genetic elements required for constituting a retroviral particle. As compared to previously described works, the present application is based in part on the use of specific types of recombinant adenovirus and/or on a specific distribution of the retroviral genetic elements. As demonstrated in the present application, the methods and constructs according to the invention make it possible to obtain high levels of retroviral particle production, are effective, and display increased safety in vitro, ex vivo as well as in vivo. Furthermore, the defective recombinant retroviruses so produced are infectious, and are capable of transferring a nucleic acid into a cell with high efficiency.
A first subject of the invention therefore concerns a composition comprising the whole set of genetic elements required for constituting a retroviral particle, incorporated in one or several recombinant adenoviruses defective for all or part of the E1 and E4 regions at least.
In the context of the invention, the term “genetic elements required for constituting a retroviral particle” refers to the whole set of nucleic acid sequences, coding and regulatory, which in cis or in trans are necessary and sufficient to constitute a retroviral particle, that is, a physical particle expressing the retroviral envelope at its surface, and inside of which are found the different proteins required to carry out a retroviral replication cycle, and a genome in a form which allows it to be reverse transcribed.
The organization of the retroviral genome is well understood, and comprises primarily the following elements:
An LTR region located at each end of the retroviral genome, serving in particular as origin of transcription and transcriptional promoter. This LTR region more specifically contains elements designated U3, R and U5 (see
FIG. 5
, for example). The U5 sequence, together with the U3 sequence, plays a key role during provirus integration.
A packaging sequence (Psi or &psgr;), involved in packaging the retroviral genome in the viral particle. The packaging sequence may further contain a region extending to certain elements of the gag gene, which have been reported to improve packaging efficiency.
Three coding regions, named gag, pol and env, coding for the core proteins (gag), enzymes (reverse transcriptase, protease, integrase), and the retroviral envelope (env), respectively.
To constitute a recombinant retroviral particle, a retroviral vector is generally constructed comprising genetic elements acting in cis, i.e., the LTR regions and the packaging sequence, and in which all or part of the gag, pol and/or env coding regions (acting in trans) have been deleted. Normally, such a retroviral vector also comprises a nucleic acid of interest (transgene). The coding regions not present or inactive in the retroviral vector are provided in trans (complementation functions) so as to be able to reconstitute a retroviral particle.
In a specific embodiment of the invention, the genetic elements therefore comprise a retroviral vector and nucleic acids coding for retroviral complementation functions (that is, for functions defective in the retroviral vector, eg., gag, pol and/or env).
In a more particular embodiment of the present invention, the genetic elements comprise:
a nucleic acid coding for a retroviral gag protein,
a nucleic acid coding for a retroviral pol protein,
a nucleic acid coding for an envelope protein, for example a retroviral env protein, and
a nucleic acid comprising, between two LTR regions, a retroviral packaging sequence and a nucleic acid sequence of interest.
To carry out the present invention, the genetic elements may be derived from different types of retrovirus, such as ecotropic and/or amphotropic viruses. In particular, these may be retroviruses belonging to the family of oncoviruses, lentiviruses or spumaviruses. In the oncovirus family, particular examples include the slow oncoviruses, which do not carry an oncogene, such as for example MoMLV, ALV, BLV or MMTV, and rapid oncoviruses, such as RSV for example. In the lentivirus family, examples include HIV, SIV, FIV or CAEV.
Furthermore, the LTR region or regions used may either be complete retroviral LTR regions, or subdomains allowing reconstitution of complete LTR regions after reverse transcription. Thus, the LTR regions used may comprise deletions of domains such as U3 and U5 for example. In a specific embodiment of the invention, the retroviral vector comprises a 5&
Klatzmann David
Perricaudet Michel
Salzmann Jean-Loup
Torrent Christophe
Yeh Patrice
Aventis Pharma S.A.
Nixon & Vanderhye P.C.
Park Hankyel T.
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