Patent Application
20140356946
The present invention is in the field of recombinant vaccine technology and relates to improvements of lentiviral vectors, which can be used as therapeutic and prophylactic vaccines. The vectors containing MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequences provide improved characteristics over other vectors.
Recombinant vaccines have been developed with the progress of recombinant DNA technology, allowing the modification of viral genomes to produce modified viruses. In this manner, it has been possible to introduce genetic sequences into non-pathogenic viruses, so that they encode immunogenic proteins to be expressed in target cells upon infection, in order to develop a specific immune response in their host.
Such vaccines constitute a major advance in vaccine technology (Kutzler et al., Nat Rev Genet, 9(10): 776-788, 2008). In particular, they have the advantage over traditional vaccines of avoiding live (attenuated) virus and eliminating risks associated with the manufacture of inactivated vaccines.
Gene delivery using modified retroviruses (retroviral vectors) was introduced in the early 1980s by Mann et al. (Cell, 33(1):153-9, 1983). The most commonly used oncogenic retroviral vectors are based on the Moloney murine leukemia virus (MLV). They have a simple genome from which the polyproteins Gag, Pol and Env are produced and are required in trans for viral replication (Breckpot et al., 2007, Gene Ther, 14(11):847-62; He et al. 2007, Expert Rev vaccines, 6(6):913-24). Sequences generally required in cis are the long terminal repeats (LTRs) and its vicinity: the inverted repeats (IR or att sites) required for integration, the packaging sequence ψ, the transport RNA-binding site (primer binding site, PBS), and some additional sequences involved in reverse transcription (the repeat R within the LTRs, and the polypurine tracts, PPT, necessary for plus strand initiation). To generate replication-defective retroviral vectors, the gag, pol, and env genes are generally entirely deleted and replaced with an expression cassette.
Retroviral vectors deriving from lentivirus genomes (i.e. lentiviral vectors) have emerged as promising tools for both gene therapy and immunotherapy purposes, because they exhibit several advantages over other viral systems. In particular, lentiviral vectors themselves are not toxic and, unlike other retroviruses, lentiviruses are capable of transducing non-dividing cells, in particular dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6):913-24), allowing antigen presentation through the endogenous pathway.
Lentiviruses are linked by similarities in genetic composition, molecular mechanisms of replication and biological interactions with their hosts. They are best known as agents of slow disease syndromes that begin insidiously after prolonged periods of subclinical infection and progress slowly; thus, they are referred to as the “slow” viruses (Narayan et al., 1989, J Gen Virol, 70(7):1617-39). They have the same basic organization as all retroviruses, but are more complex due to the presence of accessory genes (e.g., vif, vpr, vpu, nef, tat, and rev), which play key roles in lentiviral replication in vivo.
Lentiviruses represent a genus of slow viruses of the Retroviridae family, which includes the human immunodeficiency viruses (HIV), the simian immunodeficiency virus (SIV), the equine infectious encephalitis virus (EIAV), the caprine arthritis encephalitis virus (CAEV), the bovine immunodeficiency virus (BIV) and the feline immunodeficiency virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate continuously at variable rates during the course of the lifelong infection. Persistent replication of the viruses in their hosts depends on their ability to circumvent host defenses.
The design of recombinant integrative lentiviral vectors is based on the separation of the cis- and trans-acting sequences of the lentivirus. Efficient transduction in non-dividing cells requires the presence of two cis-acting sequences in the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS). These lead to the formation of a triple-stranded DNA structure called the central DNA “flap”, which maximizes the efficiency of gene import into the nuclei of non-dividing cells, including dendritic cells (DCs) (Zennou et al., 2000, Cell, 101(2) 173-85; Arhel et al., 2007, EMBO J, 26(12):3025-37).
Dendritic cells are of primary importance for antigen presentation because they constitute the main class of antigen presenting cells (APCs) whose primary function is to present antigens and initiate an immune response.
To generate an immune response, antigenic proteins must be processed by cells into peptides that are displayed on the cell surface by major histocompatibility complex proteins (MHCs). Circulating APCs present the peptide-MHC complexes to T cells in the draining lymph nodes, where they interact with T cell receptors, and, in conjunction with co-stimulatory signals, activate the T cells.
A variety of studies have shown that inoculation with lentiviral vectors leads to antigen presentation by DCs and strong activation of antigen specific cytotoxic T lymphocytes (CTLs; CD8+ T cells). Therefore, lentiviral vectors have been engineered for gene transfer and immunotherapy applications.
Lentiviral vectors have been improved in their safety by removal of the LTR U3 sequence, resulting in “self-inactivating” vectors that are entirely devoid of viral promoter and enhancer sequences originally present within the LTRs.
The lentiviral particles, which contain lentiviral vectors, can be produced by recombinant technology upon transient transfection of HEK 293T human cultured cells by different DNA plasmids:
(i) a packaging plasmid, which expresses at least the Gag, Pol Rev, Tat and, in some cases, structural and enzymatic proteins necessary for the packaging of the transfer construct;
(ii) a transfer plasmid, containing an expression cassette and HIV cis-acting factors necessary for packaging, reverse transcription, and integration; and
(iii) an envelope-encoding plasmid, in most cases the glycoprotein of vesicular stomatitis virus (VSV.G), a protein that allows the formation of mixed particles (pseudotypes) that can target a wide variety of cells, especially major histocompatibility (MHC) antigen-presenting cells (APCs), including DCs.
This procedure allows obtaining transient production of lentiviral particle vectors by the transfected cells. However, the lentiviral particle vectors may also be continuously produced by cells by stably inserting the packaging genes, the proviral coding DNA, and the envelope gene into the cellular genome. This allows the continuous production of lentiviral particle vectors by the cells without the need for transient transfection. Of course, a combination of these procedures can be used, with some of the DNAs/plasmids integrated into the cellular genome and others provided by transient transfection.
Non-integrative lentiviral vectors have designed in an attempt to mitigate the risks of potential oncogenesis linked to insertional mutagenesis events, particularly for vaccination purposes.
In vaccination based on direct injection of antigen-encoding integrative lentiviral vectors, transduced cells expressing the relevant antigen become targets of the elicited immune response and are eliminated within a few days or weeks from the vaccinated organism.
In addition, deletion in the U3 region of the 3′ LTR of the viral promoter and enhancer sequences in self-inactivating lentiviral vectors limits the likelihood of endogenous promoter activation. This deletion directly addresses the experiences gained from the SCID-X1 gene therapy trial carried out in 1998-1999, performed with Moloney virus-based retroviral vectors on children suffering from a rare form of X-linked (SCID-X1 gene) severe immunodeficiency disease (Cavazzana-Calvo et al., 2000, Science., 288(5466):669-72). During this trial, four of nine children developed leukemia as a result of the integration of the Moloney-derived retroviral vector at close proximity to the human LM02 proto-oncogene (Hacein-Bey-Abina et al., 2008, J. Clin. Invest., 118(9):3132-3142). It was demonstrated that malignancy was the consequence of the proximity of the viral U3 promoter/enhancer to the LM02 proto-oncogene.
Enhancers are cis-acting sequences, which can act as transcriptional activators at a distance. They have been widely employed in viral derived vectors because they appear to be the most efficient for obtaining transgene strong expression in a variety of cell types, in particular DCs (Chinnasamy, Chinnasamy et al., 2000, Hum Gene Ther 11(13):1901-9; Rouas et al., 2008, Cancer Gene Ther 9(9):715-24; Kimura et al., 2007, Mol Ther 15(7):1390-9; Gruh et al., 2008, J Gene Med 10(1) 21-32). However, given the safety issue of insertional mutagenesis, such transcriptional enhancer sequences should be deleted from the lentiviral vector constructs to abolish the risk of insertional mutagenesis by enhancer proximity effect. This enhancer proximity effect is by far the most frequent mechanism of insertional mutagenesis and is the only effect described in human or animal cases of tumorigenic events after gene transfer.
Thus, there is a need to develop retroviral, particularly lentiviral vectors, which do not include viral enhancers and which allow sufficient expression of transgenes encoding immunogenic peptides, if possible as much expression as that observed when using the CMV promoter. Particularly, there is a need for vectors with improved titers.
A study has reported on the replacement of viral promoters by DC-specific promoters deriving from major histocompatibility complex class II genes (MHC class II) (Kimura et al., 2007, Mol Ther 15(7):1390-9) and dectin-2 genes (Lopes et al., 2008, J Virol β2(1):86-95). The dectin-2 gene promoter used in Lopes et al. contains a putative enhancer and an adenoviral conserved sequence (inverted terminal repeats in adenovirus promoter) (Bonkabara et al., 2001, J. Immunology, 167:6893-6900). The MHC class II gene promoter used by Kimura et al. does not contain any known enhancer.
Yet, without an enhancer, the MHC class II promoter was found not to provide sufficient transgene expression in DCs. In particular, lentiviral vectors including MHC class II promoters did not provoke an immune reaction in immunocompetent C57BL/6 mice, in contrast to the immune responses observed with CMV promoters/enhancers. Although integration and persistent transgene expression were observed after injection in mice, the lentiviral vectors transcribed through MHC class II promoters failed to stimulate an antigen-specific CD8+ cytotoxic T-lymphocyte response, even after vaccination boost. The authors of these studies therefore concluded that the use of MHC class II promoters was of interest only for applications where persistence of expression is sought as in gene replacement therapy, but not in the context of immunotherapy.
Thus, the MHC class II promoter is not an adequate promoter for lentiviral vectors for induction of an immune response against an antigen. Moreover, the dectin-2 promoter is dendritic cell specific, which does not allow elimination of vectors that are integrated into other non-expressing cell types. Moreover, the dectin-2 promoter appears to contain an enhancer. Thus, the dectin-2 promoter is not a good promoter for lentiviral vectors for safety reasons.
Preferably, in immunotherapy, lentiviral vectors provide effective expression of the transgene that elicits a desired specific immune response. This requires that the expression is at a high level in APCs, such as dendritic cells.
It is also preferable that the cells transduced by the lentiviral vectors are eliminated by the immune response to provide a higher degree of safety. That is, the immune response generated against the transgene can elicit an immune response in the host sufficient to eliminate the cells that are transduced by the lentiviral vectors. The elimination of transduced cells eliminates the persistence of the lentiviral vector in the host, and possible secondary effects of the vector. In order for the transduced cells to be eliminated, expression is required in non-dendritic cells at a level that allows elimination by the immune response.
At the same time, the promoter should maximize immune stimulation through the key cells (i.e., dendritic cells) involved in the activation of naïve and memory T cells, and should minimize the risk of insertional mutagenesis and genotoxicity in stem cells, leading to malignancies. Thus, the promoter should have sufficiently high activity in dendritic and other cells, but not contain an enhancer. Based on these criteria, viral promoters, such as the CMV promoter, are not ideal because of the presence of strong enhancers. These criteria are summarized as follows:
The vector should be capable of being generated at high titers to maximize delivery and expression, while minimizing contaminants.
Thus, a need exists in the art for improved vectors. The present invention fulfils these needs in the art.
The invention encompasses compositions comprising lentiviral vectors and methods of making and using the vectors. In one embodiment, the invention encompasses a lentiviral vector comprising an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence, preferably further comprising an MHC class I or β2 microglobulin promoter.
The invention encompasses methods for producing a lentiviral vector comprising inserting at least 300 nucleotides, preferably 300-400, 300-600 or 300-1100 nucleotides, of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence into a lentiviral vector.
Preferably, the upstream promoter sequence is inserted upstream of an MHC class I or β2 microglobulin promoter. Most preferably, the upstream promoter sequence is inserted in the same orientation as the MHC class I or β2 microglobulin promoter.
In some embodiments, the upstream promoter sequence is an MHC class I upstream promoter sequence. In some embodiments, the upstream promoter sequence is a β2 microglobulin upstream promoter sequence, preferably comprising SEQ ID NO:1 or SEQ ID NO:27.
In some embodiments, the promoter is an MHC class I promoter. In some embodiments, the promoter is a β2 microglobulin promoter.
The invention encompasses a lentiviral vector comprising at least 300 nucleotides, preferably 300-400, 300-600 or 300-1100 nucleotides, of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence.
Preferably, the upstream promoter sequence is upstream of an MHC class I or β2 microglobulin promoter. Most preferably, the upstream promoter sequence is in the same orientation as the MHC class I or β2 microglobulin promoter.
In some embodiments, the upstream promoter sequence is an MHC class I upstream promoter sequence. In some embodiments, the upstream promoter sequence is a β2 microglobulin upstream promoter sequence, preferably comprising SEQ ID NO:1 or SEQ ID NO:27. In some embodiments, the promoter is an MHC class I promoter. In some embodiments, the promoter is a β2 microglobulin promoter.
Preferably, the MHC class I promoter is an HLA-A2 promoter, HLA-B7 promoter, or an HLA-E promoter.
Preferably, the upstream promoter sequence is an HLA-A2, HLA-B7, or an HLA-E, or HLA-DRα upstream promoter sequence.
Preferably, the lentiviral vector comprises a lentiviral cPPT/CTS sequence. Preferably, the lentiviral vector comprises a lentiviral ψ (psi) sequence.
The invention encompasses an isolated host cell comprising a lentiviral vector of the invention. The invention encompasses a lentiviral vector of the invention for use as a medicament or vaccine, particularly for gene therapy.
In preferred embodiments, the upstream promoter sequence comprises the nucleotide sequence of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.
The effect of MHC class I, MHC class II, or β2 microglobulin upstream promoter sequences on lentiviral vector titers was examined. The upstream promoter sequences are located upstream of the Ets/ISRE and NF-Kb binding sites found in the β2 microglobulin and MHC class I promoters (
The upstream promoter sequences of β2m and MHC Class I promoters show some similarity at the nucleotide level (
First, an upstream promoter sequence of β2m was inserted into a lentiviral vector upstream of and in the same orientation as the β2m and MHCI promoters HLA-A2, HLA-B7, and HLA-E. For comparison, the upstream promoter sequence of β2m was inserted into lentiviral vectors upstream of and in the same orientation as the Ubiquitin (UBC) gene promoter, the CMV promoter, or an MHCII promoter (HLA-DRα). In these vectors, the promoters drive expression of green fluorescent protein (GFP).
To look for expression, the vectors were packaged by cotransfection in HEK-293T cells with an encapsidation plasmid and a plasmid providing VSV.G envelope, essentially as described in Naldini et al, 1996, Science 272:263-7. HEK-293T cells were then transduced with particles of the different vectors. Expression was detected in the cells with all vectors.
The addition of the β2m upstream promoter sequence into lentiviral vectors with a β2m, HLA-A2, HLA-B7, or HLA-E promoter resulted in an approximately 2-5 fold increase in viral titers. In contrast, addition of the β2m upstream promoter sequence into lentiviral vectors with a CMV promoter, an UBC promoter, or an HLA-A2 promoter demonstrated little effect on the titers (
Next, the upstream promoter sequence of β2m was inserted into a lentiviral vector upstream of and in the reverse orientation as the β2m and MHCI promoters HLA-A2, HLA-B7, and HLA-E. For comparison, the upstream promoter sequence of β2m was inserted into lentiviral vectors upstream of and in the reverse orientation as the Ubiquitin (UBC) genes promoter, the CMV promoter, or an MHCII promoter (HLA-DRα). The addition of the β2m upstream promoter sequence into lentiviral vectors upstream of and in the reverse orientation as the HLA-A2, HLA-B7, or HLA-E promoter did not result in an increase in viral titers (
Next, the upstream promoter sequence of β2m was inserted into a lentiviral vector downstream of the transgene and in the same or reverse orientation as the β2m and MHCI promoters HLA-A2, HLA-B7, and HLA-E. For comparison, the upstream promoter sequence of β2m was inserted into lentiviral vectors downstream of the transgene and in the same or reverse orientation as the Ubiquitin (UBC) genes promoter, the CMV promoter, or an MHCII promoter (HLA-DRα). The addition of the β2m upstream promoter sequence downstream of the transgene and in the same orientation as the β2m, HLA-A2, HLA-B7, or HLA-E promoter resulted in only a small increase in viral titers (
Next, the upstream promoter sequence of β2m was inserted into a lentiviral vector outside of the LTR-LTR region in the same or reverse orientation as the β2m and MHCI promoters HLA-A2, HLA-B7, and HLA-E. For comparison, the upstream promoter sequence of β2m was inserted into lentiviral vectors outside of the LTR-LTR region in the same or reverse orientation as the Ubiquitin (UBC) genes promoter, the CMV promoter, or an MHCII promoter (HLA-DRα). The addition of the β2m upstream promoter sequence outside of the LTR-LTR region in the same or reverse orientation resulted in no apparent difference in viral titers (
Next, an upstream promoter sequence of HLA-E was inserted into a lentiviral vector upstream of and in the same or reverse orientation as the β2m and MHCI promoters HLA-A2, HLA-B7, and HLA-E. For comparison, the upstream promoter sequence of HLA-E was inserted into lentiviral vectors upstream of and in the same or reverse orientation as the Ubiquitin (UBC) genes promoter, the CMV promoter, or an MHCII promoter (HLA-DRα).
The addition of the HLA-E upstream promoter sequence into lentiviral vectors upstream of the β2m, HLA-A2, HLA-B7, or HLA-E promoter resulted in an approximately 2-4 fold increase in viral titers (
An upstream promoter sequence of HLA-A2 was inserted into a lentiviral vector upstream of and in the same or reverse orientation as the β2m promoter. The addition of the HLA-A2 upstream promoter sequence into lentiviral vectors upstream of the β2m promoter resulted in an approximately 3-4 fold increase in viral titers in both orientations.
An upstream promoter sequence of HLA-B7 was inserted into a lentiviral vector upstream of and in the same or reverse orientation as the β2m promoter. The addition of the HLA-B7 upstream promoter sequence into lentiviral vectors upstream of the β2m promoter resulted in an approximately 10 fold increase in viral titers in the same orientation and an approximately 4 fold increase in viral titers in the reverse orientation.
An upstream promoter sequence of HLA-DRα was inserted into a lentiviral vector upstream of and in the same or reverse orientation as the β2m promoter. The addition of the HLA-DRα upstream promoter sequence into lentiviral vectors upstream of the β2m promoter resulted in an approximately 6 fold increase in viral titers in the same orientation and an approximately 4 fold increase in viral titers in the reverse orientation.
Since the upstream sequences were all of about 300-400 nt in size, the effect of larger upstream sequences (500-1100nt) was investigated. A larger upstream promoter sequence of β2m (1058 bp) was inserted into a lentiviral vector upstream of and in the same orientation as the β2m promoter. The larger upstream promoter sequence did not further increase viral titers, but did retain most of the increased viral titers, as compared to the smaller (330 bp) upstream sequence (
A larger upstream promoter sequence of HLA-A2 (531 bp) was inserted into a lentiviral vector upstream of and in the same orientation as the HLA-A2 promoter. In this case, the larger HLA-A2 upstream promoter sequence increased viral titers 3 fold; whereas, the smaller (322 bp) HLA-A2 upstream sequence had a negative effect on the HLA-A2 promoter (
A larger upstream promoter sequence of HLA-B7 (511 bp) was inserted into a lentiviral vector upstream of and in the same orientation as the HLA-B7 promoter. In this case, the larger HLA-B7 upstream promoter sequence further increased viral titers 2-3 fold as compared to the smaller (352 bp) HLA-B7 upstream sequence (
A larger upstream promoter sequence of HLA-DRα (522 bp) was inserted into a lentiviral vector upstream of and in the same orientation as the HLA-DRα promoter. In this case, the larger HLA-DRα upstream promoter sequence had the same effect on the viral titers as compared to the smaller (356 bp) HLA-DRα upstream sequence (
The effect of the insertion of multiple upstream promoter sequences was investigated. The HLA-E upstream promoter sequence was inserted into a lentiviral vector upstream, downstream, and both upstream and downstream of the β2m promoter. While insertion upstream resulted in a 3-4 fold increase in titers, insertion downstream had no effect on titers and insertion both upstream and downstream resulted in decrease in viral titers.
The present invention has thus, as a main object, a lentiviral vector comprising a β2m or MHCI or MHCII upstream promoter sequence, and methods for making and using such a vector.
Lentiviral Vector
Within the context of this invention, a “lentiviral vector” means a non-replicating vector for the transduction of a host cell with a transgene comprising cis-acting lentiviral RNA or DNA sequences, and requiring lentiviral proteins (e.g., Gag, Pol, and/or Env) that are provided in trans. The lentiviral vector contains cis-acting packaging sequences, but lacks expression of functional Gag, Pol, and Env proteins. The lentiviral vector may be present in the form of an RNA or DNA molecule, depending on the stage of production or development of said retroviral vectors.
The lentiviral vector can be in the form of a recombinant DNA molecule, such as a plasmid. The lentiviral vector can be in the form of a lentiviral particle vector, such as an RNA molecule(s) within a complex of lentiviral and other proteins. Typically, lentiviral particle vectors, which correspond to modified or recombinant lentivirus particles, comprise a genome which is composed of two copies of single-stranded RNA. These RNA sequences can be obtained by transcription from a double-stranded DNA sequence inserted into a host cell genome (proviral vector DNA) or can be obtained from the transient expression of plasmid DNA (plasmid vector DNA) in a transformed host cell.
Lentiviral vectors derive from lentiviruses, in particular human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV), which are modified to remove genetic determinants involved in pathogenicity and introduce new determinants useful for obtaining therapeutic effects.
Such vectors are based on the separation of the cis- and trans-acting sequences. In order to generate replication-defective vectors, the trans-acting sequences (e.g., gag, pol, tat, rev, and env genes) can be deleted and replaced by an expression cassette encoding a transgene.
Efficient integration and replication in non-dividing cells generally requires the presence of two cis-acting sequences at the center of the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS). These lead to the formation of a triple-stranded DNA structure called the central DNA “flap”, which acts as a signal for uncoating of the pre-integration complex at the nuclear pore and efficient importation of the expression cassette into the nucleus of non-dividing cells, such as dendritic cells.
In one embodiment, the invention encompasses a lentiviral vector comprising a central polypurine tract and central termination sequence referred to as cPPT/CTS sequence as described, in particular, in the European patent application EP 2 169 073.
Further sequences are usually present in cis, such as the long terminal repeats (LTRs) that are involved in integration of the vector proviral DNA sequence into a host cell genome. Vectors may be obtained by mutating the LTR sequences, for instance, in domain U3 of said LTR (ΔU3) (Miyoshi H et al, 1998, J Virol. 72(10):8150-7; Zufferey et al., 1998, J Virol 72(12):9873-80).
In one embodiment, the invention encompasses a lentiviral vector comprising LTR sequences, preferably with a mutated U3 region (ΔU3) removing promoter and enhancer sequences in the 3′ LTR.
The packaging sequence ψ (psi) is incorporated to support encapsidation of the polynucleotide sequence into the vector particles (Kessler et al., 2007, Leukemia, 21(9):1859-74; Paschen et al., 2004, Cancer Immunol lmmunother 12(6):196-203).
In one embodiment, the invention encompasses a lentiviral vector comprising a lentiviral packaging sequence ψ (psi) and an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence.
Further additional functional sequences, such as a transport RNA-binding site or primer binding site (PBS) or a Woodchuck PostRegulation Element (WPRE), can also be advantageously included in the lentiviral vector polynucleotide sequence of the present invention, to obtain a more stable expression of the transgene in vivo.
In one embodiment, the invention encompasses a lentiviral vector comprising a PBS. In one embodiment, the invention encompasses a lentiviral vector comprising a WPRE and/or an IRES.
Thus, in a preferred embodiment, the lentiviral vector comprises an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence, at least one cPPT/CTS sequence, one ψ sequence, one (preferably 2) LTR sequence, and an expression cassette including a transgene under the transcriptional control of a promoter, particularly an MHC class I or β2 microglobulin promoter.
Transgene
The invention encompasses a lentiviral vector containing a transgene. Within the context of this invention, a “transgene” is a nucleic acid sequence within a lentiviral vector that is not normally present in a cell to be transduced with the lentiviral vector. The lentiviral vector serves to introduce this sequence into the transduced cell. The term “transgene” does not include those sequences of the vector that facilitate transduction of the transgene. The transgene may be a nucleic acid sequence from another organism. Alternatively, the transgene may be a nucleic acid sequence from the same organism, but having different regulatory sequences controlling its expression. The transgene may be a sense or antisense nucleic acid molecule. According to a preferred embodiment of the invention, the transgene sequence encodes an immunogenic polypeptide.
Preferably, the immunogenic polypeptide is viral, parasitic, bacterial, or fungal. In one embodiment, the immunogenic polypeptide is a tumor antigen.
This immunogenic polypeptide preferably comprises one or several epitope(s) from agents of infectious diseases, for example antigen(s) from Gag, Pol, and/or Nef proteins of HIV.
Several epitopes forming a polyepitope may also be encoded by the transgene of the invention.
In a particular embodiment, such epitope is derived from target antigens identified in tumors, and can be chosen in such a way that a cell-mediated immune response is obtained against it. Target antigens are well documented in the art, which can be selected with respect to several types of tumors and in particular in melanomas or in carcinomas, including renal carcinomas, bladder carcinomas, colon carcinomas, lung carcinomas, breast cancers, leukemias, and lymphomas.
B2M and MHCI Promoters
The invention encompasses the insertion of a β2m or MHC Class I (MHCI) promoter into a lentiviral vector. As used herein, an “MHC Class I (MHCI) promoter” includes a naturally occurring or synthetic MHC Class I promoter. The term “MHC Class I promoter” does not include a β2m promoter.
Naturally Occurring MHCI and a β2m Promoters
Examples of naturally occurring MHCI promoters are the HLA-A2, HLA-B7, HLA-Cw5, HLA-E, HLA-G gene promoters. These naturally occurring MHCI promoters are generally cloned or reproduced from the promoter region of a gene encoding the MHC class I protein, or referred to as putatively encoding such proteins in genome databases (ex: NCBI polynucleotide database http://www.ncbi.nlm.nih.gov/guide/dna-rna). Both β2m and class I MHC proteins enter the Major Histocompatibility Complex (MHC). Preferred promoters are set forth in U.S. Patent Publn. 2014/0120132-A1, which are hereby incorporated by reference.
The proteins encoded by these genes are found in almost all cell types. MHCI proteins are generally present at the surface of the membrane of leucocytes, where they are associated with the β2-microglobulin (β2m) protein. The role of these associated proteins is to present peptides from endogenous sources to CD8+ T cells. They thus play a central role to the generation of the antigen-specific immune response. Because β2m and MHC proteins have been widely studied and described for many years, their genes are well characterized and detectable using sequence comparison tools, such as the BLAST method (Altschul, S. F. et al. (1990). Basic local alignment search tool. J. Mol. Biol. 215(3):403-410).
β2m and MHC class I promoters share the ability to be strongly activated in dendritic cells, as well as, to lower intensity, in the majority of the other human body tissues.
The β2m and MHC class I promoters of the invention can contain further regulatory elements, such as one or more Sp1 and ETs binding sites. In a preferred embodiment, the MHC class I promoter contains 2 Sp1 binding sites and 1 Ets binding site. In other embodiments, Ap1 and/or Ap2 sites are further contained in the MHC class I promoter.
Preferred MHC class I promoters are human HLA-A2, HLA-B7, HLA-Cw5, HLA-E, HLA-F, and HLA-G promoters.
Synthetic β2m and MHC Class I Promoters
β2m and MHC class I promoters can also be synthetic. Synthetic β2m and MHC class I promoters include promoters that are synthesized using molecular biological techniques to assemble the individual components of an β2m and MHC class I promoter or that are derived from naturally occurring β2m and MHC class I promoters using molecular biological techniques.
ISRE
The transcription of β2m and MHC class genes is usually mediated by two major regulatory elements: Interferon stimulated response element (ISRE) and the SXY module (encompassing the W/S, X1X2/Site α and Y/enhancer B regulatory elements) (see
These regulatory promoter elements are localized in a region extending approximately from nucleotides −220 to −95 upstream of the transcription initiation site. They mediate tissue-specific and cytokine-induced transcription of β2m and MHC class I genes.
The ISRE of β2m and MHC class I gene promoters generally contains binding sites for interferon regulatory factor (IRF) family members. It is thus a property of MHC class I promoters to bind to interferon regulatory factor (IRF) family members. This may be verified, for example, by gel shift assays.
NF-κB Binding Site
Another regulatory element, the enhancer A (containing binding sites for nuclear transcription factor κB (NF-κB)) is present in most cases. It is thus a property of β2m and MHC class I promoters to bind to nuclear transcription factor κB (NF-κB). This may be verified, for example, by gel shift assays.
SXY Module
In addition to ISRE, β2m and MHC class I promoters generally share another set of conserved upstream sequence motifs, consisting of four regulatory elements: the S or W box, the X1/CREX2 boxes or site α, and the Y box or enhancer B, which together are termed the SXY module. This SXY module is generally cooperatively bound by a multiprotein complex containing regulatory factor X (RFX; consisting of RFX5, RFXB/ANK and RFXAP), cAMP response element binding protein (CREB)/activating transcription factor (ATF), and nuclear factor Y (NFY), which acts as an enhanceosome driving transactivation of these genes. It is thus a property of β2m and MHC class I promoters to bind to these factors. This may be verified, for example, by gel shift assays.
In contrast, MHC class II promoters do not display enhancer A, nor ISRE, elements (Van den Elsen, P. J. et al, 1998, Immunogenetics. 48:208-221). Furthermore, RFX and CIITA in MHC class II gene regulation have been found of crucial importance as illustrated by studies with cell lines established from patients with the bare lymphocyte syndrome (BLS), a severe combined immunodeficiency due to mutations in one of the RFX subunits or CIITA (DeSandro, A. et al., 1999, Am J Hum Genet, 65:279-286). Also, lack of either CIITA or one of the RFX subunits affects the functioning and assembly of the MHC enhanceosome, respectively, leading to a lack of MHC class II and reduced levels of MHC class I transcription (Van den Elsen, P. J. et al. 2004, Current Opinion in Immunology, 16:67-75).
β2M and MHCI and MHCII Upstream Promoter Sequences
The invention encompasses the insertion of a 132m, MHC Class I (MHCI), or MHC Class II (MHCII) upstream promoter sequence into a lentiviral vector. As used herein, a “β2m upstream promoter sequence” refers to 1100 base pairs or less of the sequences found immediately upstream of the Ets/ISRE binding site in the naturally occurring β2 microglobulin promoter, as illustrated in
In various embodiments, the upstream promoter sequence comprises less than 1100, 1000, 900, 800, 700, 600, 550, 500, 450, 400, or 350 nucleotides of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence
In various embodiments, the upstream promoter sequence comprises at least 300, 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence.
In various embodiments, the upstream promoter sequence comprises 300, 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, or 500 to 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, 550, 600, 700, 800, 900, 1000, or 1100 nucleotides (in all possible combinations of ranges) of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence. Preferably, the upstream promoter sequence comprises 300-400, 300-500, 300-600, 300-700, or 300-1100 nucleotides of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence. Most preferably, the upstream promoter sequence comprises 300-335 nucleotides of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence.
Preferably, the B2M upstream promoter sequence comprises the nucleotide sequence:
AGAAGTTCTCCTTCTGCTAGGTAGCATTCAAAGATCTTAATCTTCTGGGTT TCCGTTTTCTCGAATGAAAAATGCAGGTCCGAGCAGTTAACTGGCGGGGGCACC ATTAGCAAGTCACTTAGCATCTCTGGGGCCAGTCTGCAAAGCGAGGGGGCAGC CTTAATGTGCCTCCAGCCTGAAGTCCTAGAATGAGCGCCCGGTGTCCCAAGCTG GGGCGCGCACCCCAGATCGGAGGGCGCCGATGTACAGACAGCAAACTCACCC AGTCTAGTGCATGCCTTCTTAAACATCACGAGACTC (SEQ ID NO:1). Preferably, the B2M upstream promoter sequence comprises the nucleotide sequence SEQ ID NO:27 or 28.
Preferably, the MHC Class I upstream promoter sequence comprises an HLA-A2, HLA-B7, HLA-Cw5, HLA-E, or HLA-G upstream promoter sequence.
Preferably, the MHC Class I upstream promoter sequence comprises the nucleotide sequence of any of SEQ ID NOs:2-7 or SEQ ID NO:29-34.
Preferably, the MHC Class I upstream promoter sequence comprises the nucleotide sequence:
Preferred upstream promoter sequences include the following:
In some embodiments, the vectors comprise any of SEQ ID Nos. 37-46.
Production of Lentiviral Vectors
In one embodiment, the invention encompasses a method comprising inserting an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence into a lentiviral vector. The method can further comprise inserting any of the other nucleic acid elements mentioned herein, such as a DNA flap sequence.
The invention encompasses methods for producing a lentiviral vector comprising inserting at least 300, 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence into a lentiviral vector.
In various embodiments, the upstream promoter sequence comprises less than 1100, 1000, 900, 800, 700, 600, 550, 500, 450, 400, or 350 nucleotides of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence.
The invention encompasses methods for producing a lentiviral vector comprising inserting 300, 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, or 500 to 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, 550, 600, 700, 800, 900, 1000, or 1100 nucleotides (in all possible combinations of ranges) of an MHC class I, MHC class II, or β2 microglobulin upstream promoter sequence into a lentiviral vector. Preferably, the upstream promoter sequence comprises 300-400, 300-500, 300-600, 300-700, or 300-1100 nucleotides of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence.
Most preferably, the upstream promoter sequence comprises 300-335 nucleotides of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence.
Preferably, the MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence is inserted into a lentiviral vector comprising a MHC class I or β2 microglobulin promoter. The upstream promoter sequence can be in the same or reverse orientation as the promoter.
The invention encompasses methods for producing a lentiviral vector comprising inserting at least 300, 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence and an MHC class I or β2 microglobulin promoter into a lentiviral vector.
In various embodiments, the upstream promoter sequence comprises less than 1100, 1000, 900, 800, 700, 600, 550, 500, 450, 400, or 350 nucleotides of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence.
The invention encompasses methods for producing a lentiviral vector comprising inserting 300, 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, or 500 to 305, 310, 315, 320, 325, 330, 335, 350, 357, 400, 450, 550, 600, 700, 800, 900, 1000, or 1100 nucleotides (in all possible combinations of ranges) of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence and an MHC class I or β2 microglobulin promoter into a lentiviral vector. Preferably, the upstream promoter sequence comprises 300-400, 300-500, 300-600, 300-700, or 300-1100 nucleotides of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence. Most preferably, the upstream promoter sequence comprises 300-335 nucleotides of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence.
In one embodiment, the MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence is inserted into the lentiviral vector prior to insertion of a MHC class I or β2 microglobulin promoter.
In one embodiment, the MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence is inserted into the lentiviral vector after insertion of a MHC class I or β2 microglobulin promoter.
In one embodiment, the MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence and an MHC class I or β2 microglobulin promoter are inserted together into the lentiviral vector.
In one embodiment, a β2 microglobulin upstream promoter sequence is inserted upstream in the same orientation as an MHC class I or β2 microglobulin promoter.
In one embodiment, an MHC class I or MHC Class II upstream promoter sequence is inserted upstream in the same orientation as an MHC class I or β2 microglobulin promoter.
Preferably, the upstream promoter sequence comprises an β2 microglobulin, HLA-A2, HLA-B7, HLA-Cw5, HLA-E, or HLA-G upstream promoter sequence and the promoter is a β2 microglobulin, HLA-A2, HLA-B7, HLA-Cw5, HLA-E, or HLA-G promoter. All combinations individually are considered part of the invention.
Preferably the upstream promoter sequence comprises a nucleotide sequence comprising any of SEQ ID NO:1-SEQ ID NO:7 or SEQ ID NO:27-SEQ ID NO:36.
Production of Lentiviral Particle Vector
The present invention provides a method for producing a lentiviral particle vector, which contains an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence. Thus, the invention encompasses a lentiviral particle vector comprising an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence. A lentiviral particle vector (or lentiviral vector particle) comprises a lentiviral vector in association with viral proteins.
The insertion of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence can increase the titer of the vector.
According to one embodiment of this method, the particle vector is obtained in a host cell transformed with a DNA plasmid.
Such a DNA plasmid can comprise:
The invention allows the production of recombinant vector particles, comprising the following steps of:
i) transfecting or transducing a suitable host cell with a lentiviral vector;
ii) transfecting or transducing said host cell with a packaging plasmid vector, containing viral DNA sequences encoding at least structural and integrase proteins of a retrovirus (preferably lentivirus); Such packaging plasmids are described in the art (Dull et al., 1998, J Virol, 72(11):8463-71; Zufferey et al., 1998, J Virol 72(12):9873-80).
iii) culturing said transfected host cell in order to obtain expression and packaging of said lentiviral vector into lentiviral vector particles; and
iv) harvesting the lentiviral vector particles resulting from the expression and packaging of step iii) in said cultured host cells.
The host cell transfected or transduced with a packaging plasmid may be a stable packaging cell line. Thus, the method can comprise:
i) transfecting or transducing a packaging cell line with a lentiviral vector;
ii) culturing the cell line in order to obtain expression and packaging of said lentiviral vector into lentiviral vector particles; and
iii) harvesting the lentiviral vector particles resulting from the expression and packaging of step ii) in the cultured cell line.
For different reasons, it may be helpful to pseudotype the obtained retroviral particles, i.e. to add or replace specific particle envelope proteins. For instance, this may be advantageous to have different envelope proteins in order to distinguish the recombinant particle from natural particles or from other recombinant particles. In matter of vaccination strategy, pseudotyped particle vectors are more likely to escape the immune system, when a patient has already developed immunity against lentiviruses. This is particularly helpful when successive injections of similar particle vectors are required for immunizing a patient against a disease.
In order to pseudotype the retroviral particles of the invention, the host cell can be further transfected with one or several envelope DNA plasmid(s) encoding viral envelope protein(s), preferably a VSV-G envelope protein.
An appropriate host cell is preferably a human cultured cell line as, for example, a HEK cell line.
The method for producing the vector particle is carried out in a host cell, which genome has been stably transformed with one or more of the following components: a lentiviral vector DNA sequence, the packaging genes, and the envelope gene. Such a DNA sequence may be regarded as being similar to a proviral vector according to the invention, comprising an additional promoter to allow the transcription of the vector sequence and improve the particle production rate.
In a preferred embodiment, the host cell is further modified to be able to produce viral particle in a culture medium in a continuous manner, without the entire cells swelling or dying. One may refer to Strang et al., 2005, J Virol 79(3):1165-71; Relander et al., 2005, Mol Ther 11(3):452-9; Stewart et al., 2009, Gene Ther, 16(6):805-14; and Stuart et al., 2011, Hum gene Ther (in press), with respect to such techniques for producing viral particles.
An object of the present invention consists of a host cell transformed with a lentiviral particle vector.
The lentiviral particle vectors can comprise the following elements, as previously defined:
and optionally one of the additional elements described above.
Methods for Expressing a Transgene in a Cell
The present invention encompasses methods for expressing a transgene in a cell, preferably a non-dividing cell. The method comprises transducing a cell with a lentiviral vector or lentiviral particle vector of the invention under conditions that allow the expression of the transgene.
The cells are preferably mammalian cells, particularly human cells. Particularly preferred are human non-dividing cells.
The transgene preferably encodes an immunogenic polypeptide. The method can further comprise harvesting or isolating the polypeptide.
The lentiviral vector or lentiviral particle vector preferably comprises an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence. Preferably the vector further comprises an MHC class I or β2 microglobulin promoter.
In one embodiment, the invention encompasses a method for expressing a transgene comprising inserting an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence into a lentiviral vector and transducing a cell with the vector containing the MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence.
Therapeutic Use of Lentiviral Vectors
The present invention further relates to the use of the lentiviral vectors according to the invention, especially in the form of lentiviral particle vectors, for the preparation of therapeutic compositions or vaccines which are capable of inducing or contributing to the occurrence or improvement of an immunogical reaction against epitopes, more particularly those encoded by the transgene present in the vectors containing an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence.
The present invention thus provides vectors that are useful as a medicament or vaccine, particularly for gene therapy.
These vectors are preferentially used for the treatment or prophylaxis of infectious diseases, especially diseases associated with virus infection and more particularly, with retrovirus infection, such as AIDS and other immunodeficiencies.
The invention can also be used in treatment protocols against tumors and cancers and especially could be used in protocols for immunotherapy or vaccination therapy against tumors.
As the vectors of the invention more specifically target dendritic cells to obtain a cell-mediated immune response and especially the CTL response associated with the antigen expressed by the transgene in these cells, they are particularly useful as vaccines targeting slow or endogenous pathogenic microorganisms such as Mycobacteria or HIV virus.
Accordingly, the invention relates to an immunogenic composition comprising a lentiviral vector as previously defined.
The immunogenic compositions of the invention preferably contain cPPT and CTS sequences in the vector and vector particles to induce or to stimulate the nuclear import of the vector genome in the target cells.
During reverse transcription, cPPT and CTS sequences induce the formation of a three stranded DNA structure referred as DNA triplex, which stimulates the nuclear import of DNA vector sequence. Preferably, the vector comprises a transgene and regulatory signals of retrotranscription, expression and encapsidation of retroviral or retroviral-like origin, wherein the composition is capable of inducing or of stimulating a CTL (Cytotoxic T Lymphocytes) or a CD4 response against one or several epitopes encoded by the transgene sequence present in the vector.
The titer of the lentiviral vector is improved by inclusion of an MHC class I, MHC Class II, or β2 microglobulin upstream promoter sequence in the vector.
Thus, the lentiviral vectors according to the invention have the ability to induce, improve, or in general be associated with the occurrence of a memory CTL response. In other words, they can be used for the preparation of therapeutic composition for the treatment of tumor diseases or infectious diseases, by induction of, stimulation of, or participation in the occurrence of a cell-mediated immune response, especially a CTL response or a memory response.
The lentiviral vectors of the invention can be used in methods of treatment and methods of inducing an immune response comprising administering the lentiviral vector to a host and generating a specific immune response against the transgene in the host. The cells and antibodies generated in these hosts can be used as diagnostic reagents.
The lentiviral vectors according to the invention can be directly administered to a patient through known routes of administration, including systemic, local, or cutaneous, intramuscular, intradermal, for instance intratumoral, administration routes. Ex vivo administration, for instance ex vivo transduction of target cells followed by administration of the treated cells to the patient to be treated, is also encompassed by the invention.
In a particular embodiment, the immunogenic composition according to the invention can be directly administered to the patient, in such a way that it will induce, improve, or participate in vivo in the occurrence of a cell-mediated immune response, especially a CTL-mediated immune response.
In another embodiment, the immunogenic compositions are used once or upon repeated administrations so that they can enable the occurrence of a long-term memory cell mediated response.
The immunogenic compositions of the invention can be used to elicit or stimulate a cell-mediated immune response against multiple epitopes encoded by the nucleotides sequences of interest or transgene present in the vector or vector particles, and they can also be used to elicit or stimulate a cell-mediated immune response against the product of the entire sequence of a gene, for instance a gene of a pathogenic agent or fragments of said gene capable to encode at least 8 to 15 amino acids preferably 9 to 12 amino acids.
The invention also encompasses a lentiviral vector comprising a nucleotide sequence encoding a multiple repeat (at least 2 identical sequences) of said amino acid sequence inducing a cellular response and/or an amino acid sequence containing at least 2 different sequences corresponding to 2 epitopes of different pathogens or tumoral antigens.
As a result, the invention encompasses a composition that could be used in prophylactic and/or therapeutic vaccination protocols, for the treatment of tumors and especially as anti-cancer or anti-infectious diseases treatment.
In particular, it can be used in combination with adjuvants, other immunogenic compositions, chemotherapy, or any other therapeutic treatment.
Having thus described different embodiments of the present invention, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.
HEK 293T (human embryonic kidney cell line, ATCC CRL-11268, (Graham et al. 1977)) cells were maintained in Dubelcco's modified Eagle's medium (DMEM/High modified, Hyclone) supplemented with 10% fetal bovine serum (FBS, PAA), 1% L-Glutamine (Eurobio), 1% Penicillin-Streptomycin (Gibco by Life technologies) and 1% Sodium Pyruvate (Gibco by Life technologies).). The cell line was kept in an incubator with humidified atmosphere of 5% CO2 at 37° C.
The promoters are cloned between the MluI and BamHI sites of the pFLAP-GFP proviral plasmid.
β2m_Upstream sequence (β2m_US) cloned upstream of the promoters:
HLA-B7 and HLA-E promoters were purchased from GeneArt (Lifetechnologies), and they were designed to encompass the β2m upstream promoter sequence (β2m_US) upstream the 5′end of the original promoter sequence. To generate the HLA-B7 and HLA-E provirus plasmids, PCR reactions were performed to only amplify the wild type promoter sequences, which were cloned between the MluI and BamHI sites of the pFlap-GFP plasmid.
To add the β2m-upstream sequence (β2m_US) in the 5′ end of the HLA-A2, HLA-DRα, CMV and UBC promoters, we performed fusion PCR reactions. Briefly, three separate PCR reactions were performed: the first PCR amplify the β2m_US, the second PCR amplify the promoter, including a 25 bp overhang homologous to the end of the β2m_US. The PCR 1 and 2 products are then purified in an agarose gel (QIAquick gel extraction kit, QIAGEN) and used as matrices for the third PCR that will generate the final DNA product (β2m_US-Promoter). Primers used for the three PCR of each promoter are resumed in table 1. The PCR 3 product are gel purified and cloned in pCR®2.1-TOPO® (Life Technologies), sequenced, digested by MluI and BamHI restriction enzymes and cloned into the pFlap-GFP.
β2m_Upstream sequence cloned upstream the promoters, in reverse orientation:
The β2m upstream promoter sequence in reverse orientation (β2m_USR) was cloned upstream each promoter using fusion PCR as described above. All the USR_promoters were amplified between AscI and BamHI sites and then cloned into the pflap-GFP using MluI and BamHI sites. Primers used for the three PCR of each promoter are listed in table 2.
β2m_Upstream sequence cloning downstream the GFP in direct orientation:
β2m_US was cloned downstream the GFP reporting gene using XhoI and KpnI restriction sites. First, a PCR was performed to add the XhoI and KpnI sites in 5′ and 3′ of the β2m_US respectively. Primers used for the PCR are: forward: 5′-CTCGAGGAGAAACCCTGCAGGGAATTC-3′ (SEQ ID NO:9), reverse: 5′-GGTACCGAGTCTCGTGATGTTTAAGAAGGCA-3′ (SEQ ID NO:10). The PCR products were gel purified, cloned in pCR®2.1-TOPO® (LifeTechnologies), sequenced, digested by XhoI and KpnI restriction enzymes and cloned between the XhoI and KpnI sites of the pFlap-GFP.
β2m_Upstream sequence cloned downstream the GFP in reverse orientation:
The β2m upstream promoter sequence in reverse orientation (β2m_USR) was cloned downstream the GFP reporting gene using XhoI and KpnI restriction sites. First, a PCR was realized to add the XhoI and KpnI sites in 5′ and 3′ of the β2m_USR respectively. Primers used for the PCR are: forward: 5′-GGTACCGAGAAACCCTGCAGGGAATTCCCCAG-3′ (SEQ ID NO:11) reverse: 5′-CTCGAGGAGTCTCGTGATGTTTAAGAAGGCA-3′ (SEQ ID NO:12). The PCR product is then gel purified, cloned in pCR®2.1-TOPO® (LifeTechnologies), sequenced, digested by XhoI and KpnI restriction enzymes and cloned between the XhoI and KpnI of the pFlap.
β2m_Upstream sequence cloned between pUCori et KanR:
As the number of restrictions sites present between the pUCori and KanR sequences is restrained, we chose to clone the β2m_US into the PmlI sites. As a PmlI site is also present in the 5′LTR of the pFlap backbone, we first added the β2m_US upstream the pUCori using fusion PCR and then cloned the whole fragment between the two PmlI sites. Primers used for PCR1 (amplification of β2m_US) are: F1—5′-CACGTGGAGAAACCCTGCAGGGAATTCCCCAG-3′ (SEQ ID NO:13) and R1—5′-GAGTCTCGTGATGTTTAAGAAGGCA-3′ (SEQ ID NO:14). Primers used for PCR2 (amplification of pUCori and SV40) are: F2—5-TGCCTTCTTAAACATCACGAGACTCCTAAAACTTCATTTTTAATTT-3′ (SEQ ID NO:15), containing an overhang homologous to the end of β2m_US (in bold) and R2—5′-CACGTGATGAAATGCTAGGCGGCTGTC-3′ (SEQ ID NO:16). PCR 1 and 2 products were purified on an agarose gel and used as matrices for the third PCR, and the F1 and R2 primers were used for the amplification. The PCR 3 product are gel purified and cloned in pCR®2.1-TOPO® (Life Technologies), sequenced, digested by PmlI and cloned between the same sites in the pFlap-GFP. Cloning orientation was controlled by enzymatic digestion.
β2m_Upstream sequence cloning between pUCori et KanR, in reverse orientation:
β2m_USR was cloned between the PmlI sites as described above. Primers used for PCR1 (amplification of β2m_USR) are: F1—5′-CACGTGGAGTCTCGTGATGTTTAAGAAGGCATG-3′ (SEQ ID NO:17) and R1—5′-GAGAAACCCTGCAGGGAATTCCCCAG-3′(SEQ ID NO:18). Primers used for PCR2 (amplification of pUCori and SV40) are: F2—5-TGGGGAATTCCCTGCAGGGTTTCTCCTAAAACTTCATTTTTAATTT-3′ (SEQ ID NO:19) containing an overhang homologous to the end of β2m_USR (in bold) and R2—5′-CACGTGATGAAATGCTAGGCGGCTGTC-3′ (SEQ ID NO:20). PCR 1 and 2 products were gel purified and used as matrice for the third PCR, and the F1 and R2 primers were used for the amplification. The PCR 3 product are gel purified and cloned in pCR®2.1-TOPO® (LifeTechnologies), sequenced, digested by PmlI and cloned between the same sites in the pFlap-GFP. Cloning orientation was controlled by enzymatic digestion.
The promoters were cloned into the pFlap-GFP plasmid using the MluI and BamHI sites. As β2m and HLA-B7 promoters contain a MluI site in their sequence, an AscI site (compatible with MluI site) is used as replacement, which makes the MluI site disappear.
Short Upstream Sequences Cloned Upstream of the Promoters
HLA-A2, HLA-E and HLA-DRα short upstream sequences were purchased from GeneArt and cloned upstream their respective promoters using MluI restriction site. Orientation of the inserted sequences was controlled by sequencing. The HLA-B7 short upstream sequence was added upstream of the HLA-B7 promoter using fusion PCR Primers used for PCR1 (amplification of HLA-B7_US) are: F1—5′-GGCGCGCCCAGGTTTAAAGAGAAAACCCCTG-3′ (SEQ ID NO:17) and R1—5′-ATTGCAGACGCGGCCCTCGGAGCCTGAGA-3′ (SEQ ID NO:18). Primers used for PCR2 (amplification of HLA-B7 promoter) are: F2—5-AGGCTCCGAGGGCCGCGTCTGCAATGGGGAGGCGCACGTTGGGGATTC-3′ (SEQ ID NO:19) containing an overhang homologous to the end of HLA-B7_US (in bold) and R2—5′-CGGAAGGAAAGTGACGGGCGAA-3′ (SEQ ID NO:20). PCR 1 and 2 products were gel purified and used as matrice for the third PCR, and the F1 and R2 primers were used for the amplification. The PCR 3 product are gel purified and cloned in pCR®2.1-TOPO® (Life Technologies), sequenced, digested by MluI and BamHI restriction enzymes and cloned into the pFlap-GFP.
Long Upstream Sequences Cloned Upstream of the Promoters
HLA-A2, HLA-E and HLA-DRα long upstream sequences were purchased from GeneArt and cloned upstream their respective promoters using MluI restriction site. Orientation of the inserted sequences was controlled by sequencing. B2m_Up and HLA-B7_Up blocks (promoters+long upstream sequences) were purchased from GeneArt and cloned in the pFlap-GFP using the MluI/BamHI restrictions sites.
Double Upstream Sequences
HLA-E_US was cloned in the pFlap-ΔU3-β2m_E_US-GFP upstream of the GFP gene using XhoI and KpnI restriction sites. First, a PCR was realized to add the XhoI and KpnI sites in 5′ and 3′ of the HLA-E_US respectively. Primers used for the PCR are: forward: 5′-CTCGAGACTAATTTCTTTTTTCTTGTTGCC-3′ and reverse: 5′-GGTACCAACTTTTAGGTTTCGGTATCTCTGCACA-3. The PCR product is then gel purified, cloned in pCR®2.1-TOPO® (Life Technologies), sequenced, digested by XhoI and KpnI restriction enzymes and cloned between the same sites in the pFlap-ΔU3-β2m_E_US-GFP, allowing the obtaining of the pFlap-ΔU3-β2m_E_US-GFP_E_US.
The lentiviral vectors were produced by transient transfection of HEK 293T cells using a standard calcium phosphate precipitation protocol. HEK 293T cells were seeded at 7×106 cells in 10 cm2 Tissue Culture Dish (BD Falcon) in 10 mL of complete culture medium and maintained 24 h in an incubator with humidified atmosphere of 5% CO2 at 37° C. to adhere. For each vector produced, one tissue culture dish is transfected as following: the lentiviral backbone plasmid pFlap (10 μg), the pThV-Env1 encoding envelope plasmid (2 μg), and the pThV-GP packaging plasmid (10 μg) were mixed with 353 μL of sterile distilled water (Gibco by Life Technologies) and 125 μL of CaCl2 (Fluka). The DNA mix is then added drop to drop to 500 μL of 37° C. prewarmed HBS 2× pH=7.3 and the 1 mL of precipitate obtained was added to the culture medium of the cells. The transfected cells were then incubated at 37° C., 5% CO2. The medium was replaced 24 h after transfection by 7 mL of harvest medium without serum and the viral supernatant was harvested after an additional 24 h, clarified by centrifugation 5 min. at 2500 rpm and stored à −20° C.
For the quantification of infective particles, HEK 293T cells were seeded in 24-well plates (BD Falcon) at a density of 1×105 cells per well in complete medium containing 10% FBS and incubated for 4 h to adhere. The cells were transduced by replacing the medium with 300 μl of dilutions 1/100, 1/300 and 1/900 of viral samples in complete medium, followed by incubation at 37° C., 5% CO2 for 2 h. After adsorption, 1 mL of complete medium was added to each well. At 72 h posttransduction, the cells were trypsinized and resuspended in 300 μL of complete medium, and the percentage of cells expressing GFP was determined with a FACScalibur flow cytometer (BD Biosciences), using the FL1 channel. Two sets of three dilutions were performed for each sample tested. The values corresponding to a percentage of transduced cells less than 30% were used to calculate the approximate number of transducing units (TU) present in the viral suspension.
The quantification of total particles was performed on dilutions 10−5, 10−6 and 10−7 of each production supernatant, using a commercial kit (Perkin Elmer), following the manufacturer's recommendations.
HEK 293T cells were seeded in 6-well plates (BD Falcon) in culture medium and incubated for 4 h at 37° C., 5% CO2 in moist atmosphere. Cells were transduced with 3 successive dilutions of lentiviral vector (1/5, 1/10 and 1/20). 72 h post-incubation, cells were harvested and transduced HEK 293T cell pellets were realized. After intermediate storage at −20° C., total genomic DNA from transduced cell-pellets was extracted using a method based on QIAGEN QIAamp DNA mini kit handbook using single columns and a microcentrifuge. Extracted DNA was stored at −20° C. till used in qPCR. Quantification of the proviral DNA integrated in the host genome was performed on extracted DNA using an optimized Taqman qPCR, based on the exonuclease activity of the 5′-3′ Taq polymerase.
The probe is an oligonucleotide specific to the backbone of our lentiviral vector′ sequence. The amplification is performed with a polymerase Master Mix (Fermentas Thermo Scientific) and using Flap A primer (CCCAAGAACCCAAGGAACA) (SEQ ID N0:21), Flap S primer (AGACAA GATAGAGGAAGAGCAAAAC) (SEQ ID N0:22), and Lenti TM probe (6FAM-AACCATTAGGAGTAGCACCCACCAAGG-BBQ) (SEQ ID N0:23). In order to normalize the number of integrations to the number of cells harvested a specific amplification of cellular ACTIN gene is applied in parallel using the same Master Mix and Actine A primer (CGGTGAGGATCTTCATGAGGTAGT) (SEQ ID NO:24), Actine S primer (AACACCCCAGCCATGTACGT) (SEQ ID NO:25) and Humura ACT TM probe (6FAM-CCAGCCAGGTCCAGACGCAGGA-BBQ) (SEQ ID NO:26). Both reactions are achieved on MasterCycler Ep Realplex S (Eppendorf) following the thermal program (2 min at 50° C., 10 min at 95° C. and 40 cycles of 15 seconds at 95° C. and 1 min at 63° C.). The analysis is performed on MasterCycler Ep Realplex Software.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13305738.0 | Jun 2013 | EP | regional |
