Retroviral hybrid vectors pseudotyped with LCMV

Details Retroviral hybrid vectors pseudotyped with LCMV Retroviral hybrid vectors pseudotyped with LCMV

1999-05-11

2002-08-27

Nguyen, Dave T. (Department: 1636)

Chemistry: molecular biology and microbiology

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

C435S320100, C435S455000, C435S456000, C435S069100, C435S070100

Reexamination Certificate

active

06440730

ABSTRACT:

The present invention relates in general to the pseudotyping of retroviruses with lymphocytic choriomeningitis virus. In particular, the invention relates to pseudotyping in MLV packaging cells which are optionally env-deleted, or in packaging cells derived from lentiviruses. Preferably, pseudotyping is carried out by infection with LCMV or a preferably env-deleted mutant, or by transfection with an expression plasmid containing the gp-gene of LCMV or a part thereof and optionally, in addition, the np-, the 1- and/or the z-gene of LCMV. The invention also relates to the use of such pseudotypes for the infection of cells, particularly the use in gene therapy.
Retroviral vectors are increasingly being used in the state of the art, for example, for gene transfer in genetic engineering and medical research or in gene therapy approaches (cf. e.g. C. Baum et al. in
Seminars in Oncology: Gene Therapy of Cancer: Translational approaches from preclinical studies to clinical implementations
., eds. Gerson & Lattime, Academic Press, 1998). The retroviral vectors are mostly derived from murine leukaemia viruses (MLV) and contain all the sequences of the LTR regions required for integration and the &psgr;-element responsible for packaging. The regions coding for the virus proteins are replaced by foreign genes and the control sequences thereof which it would be desirable to introduce into human cells. The vectors are expressed in so-called helper cell lines (packaging cell lines) which contain a copy of a complete retrovirus genome. It synthesises all the proteins required for replication and infection, but is unable to package its genomic virus-RNA into particles because it has a defect in the &psgr;-sequences. If the retroviral vectors are inserted into these helper cells and transcribed, the transgenic mRNA formed is able, by means of the &psgr;-region which is characteristic of it, to interact with the structure proteins of the helper virus and be packaged to particles. The recombinant virions, which possess no genetic information at all for virus components, adsorb on cells by way of their surface proteins, the capsids are taken up in the cytoplasm, and the transgenic RNA is converted to double-stranded DNA and integrated into the host cell genome. The advantage of this system is the stable integration of the foreign genes which are passed on to the daughter cells on division. The non-specific integration at arbitrary sites of the cell genome, which is characteristic of retroviruses, is a disadvantage.
Retroviral vectors impart a stable colinear integration (i.e. without recombinations and rearrangement of the coding sequences in the vector genome) and thereby a long-term expression of the transgene. Long-term gene expression has otherwise been possible hitherto only by means of the episomal herpes virus vectors or the adeno-associated virus vectors (AAV vectors). The packaging systems (packaging cell lines) have not yet, however, been optimised for the latter vector systems. Moreover, AAV vectors have a lower packaging capacity (about 5 kb for AAV compared with about 10-12 kb for retroviral vectors).
In addition to the gene to be transferred, the transgene, packaging lines also express the vector genome which contains retroviral cis elements. The genomic vector transcript does not, therefore, code for retroviral proteins but is inserted in the packaging lines with the aid of the gag-, pol- and env-gene products into a virion which is infectious but not capable of replication. This virion may then be used as a retroviral vector for transferring the transgene integrated into the vector genome into the desired target cells without further proliferation of the vector occurring there. In other words, the viral vector is only able to infect the target cells but is unable to proliferate any further therein.
The development of retroviral packaging systems is already well advanced and vector supernatants that are free from viruses capable of replication can be produced in large quantities under GMP conditions (Good Manufacturing Practice; Directive of the Commission for laying down principles and guidelines of good manufacturing practice (GMP) for certain medicaments for use in humans (91/356/EEC) of 13.6.91). Vectors based on murine leukaemia virus (MLV vectors) have already been used repeatedly in clinical trials (P. Chu et al., J. Mol. Med. 76 (1998) 184-192).
Two fundamental types of retroviral packaging systems are known in the prior art (J. M. Wilson, Clin. Exp. Immunol. 107 Suppl. 1 (1997) 31-32; C. Baum et al. 1998), loc cit.).
MLV packaging cell lines contain the retroviral genes gag, pol and env (
FIG. 1
) and the sequences required for packaging the retroviral RNA are deleted (C. Baum et al., (1998), loc. cit.).
The second type of known packaging systems is derived from the lentiviruses (R. Carroll et al., J. Virol. 68 (1994) 6047-6051; P. Corbeau et al., Proc. Natl. Acad. Sci. USA 93 (1996) 14070-14075; L. Naldini et al., Science 272 (1996) 263-267; C. Parolin et al., J. Virol. 68 (1994) 3888-3895; J. Reiser et al., Proc. Natl. Acad. Sci. USA 93 (1996) 15266-15271; J. H. Richardson et al., J. Gen. Virol. 76 (1995) 691-696; T. Shimada et al., J. Clin. Invest. 88 (1991) 1043-1047). Lentiviruses are complex retroviruses which, in addition to the gag, pol and env gene products also express a series of regulatory genes. Examples of lentiviruses from which packaging systems were derived are the human immunodeficiency virus (HIV), the “simian immunodeficiency virus” (SIV) and the “feline immunodeficiency virus” (FIV). The structure of the lentiviral packaging systems is similar, in principle, to that of the MLV vectors.
An advantage of lentiviral vectors is that they are also able to infect resting cells. In the case of MLV vectors, on the other hand, the vector genome can be transported into the cell nucleus only during cell division, i.e. when the nuclear membrane is dissolved. However, in view of the complex structure of the lentiviral genome, packaging systems derived from lentiviruses have disadvantages which are manifested in a comparatively low titre and relatively poor stability. Due to the complex genome structure, cis and trans elements in the genome cannot be separated clearly from one another. In the packaging constructs that express lentiviral gag, pol and env genes there are also to be found, therefore, important cis-regulatory sequences (e.g. parts of the packaging signal) which must also be contained in the vector genome. Due to these homologies, recombinations between vector genome and the packaging constructs may occur and thus the release of retroviruses capable of replication (e.g. an HIV wild virus which would be highly undesirable), so these systems are not comparable with MLV packaging lines.
All the vector systems known hitherto in the prior art also have some crucial shortcomings which prevent successful use in gene therapy: 1. Retroviral vectors are mostly produced only in inadequate titres and cannot be concentrated any further due to the instability of their envelope proteins. 2. Vector particles cannot be purified without loss of infectiousness due to the instability of their envelope proteins. Such purification is essential, however, as the cell culture supernatants from which vectors are harvested are contaminated by cellular constituents. 3. Due to their envelope proteins, retroviral vectors are inactivated by human serum complement. 4. The receptor for the envelope protein of the classic amphotrophic vectors is expressed on virtually all the cell lines considered. However, many primary human cells such as hepatocytes and haematopoietic stem cells which are attractive targets of gene therapy are deficient in functional amphotrophic receptors, as a result of which transduction is rendered difficult or prevented.
The object of the present invention is, therefore, to provide retroviral packaging systems which do not have the disadvantages of the packaging cell lines known in the prior art.
In particular, the object of the present invention is to provide pac

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