# Patent

## Title
Gene transfer vectors comprising genetic insulator elements and methods to identify genetic insulator elements

## Abstract

The present invention relates to a gene transfer vector (GTV) and in particular to an integrating gene transfer vector (IGTV), which comprises at least one genetic insulator element (GIE), wherein the each comprises at least two copies of an element selected from the group consisting of: a CTF binding site; a first CTCF binding site and a second CTCF binding site, wherein the first and the second CTCF binding sites are derived from the regulatory sequences of different genes.

## Background
This application is a National Stage of PCT/IB10/000950 filed Apr. 2, 2010 and claims the benefit of 61/254,351 filed Oct. 23, 2009 and 61/166,420 filed Apr. 3, 2009.
FIELD OF THE INVENTION
The present invention relates to gene transfer vectors and in particular integrating gene transfer vectors which comprise at least one Genetic Insulator Element (GIE) which limits the effects of a regulatory sequence upon another regulatory or coding sequence disposed upon the other side of the GIE. The present invention also relates to methods of identifying GIEs and to the use of an integrating gene transfer vector, in particular a retrovirus vector, in in vivo and ex vivo gene therapy methods as well as to cells and organisms transformed using vectors according to the present invention.
INTRODUCTION
The need for better gene transfer systems, in terms of specificity, efficacy and safety has been and still remains a major challenge in the development of gene therapy, so that the risk/benefit balance of treating a condition via a gene therapy method will be improved sufficiently to allow the routine use of gene therapy in patients.
Retroviral (RV) and other viral and non-viral vectors used in gene therapy often have a preference for particular chromosomal integration regions or targets. It is also well known that chromosomal insertion of a vector can activate or indeed inactivate genes nearby on the chromosome and that chromosomal regulatory sequences can affect the expression of vector encoded, genes, this phenomenon being known as regulatory cross talk. When endogenous genes are improperly expressed in this way, this can lead to these cells becoming cancerous. This oncogenic potential of vectors may stem from the promiscuous activation of cellular genes by endogenous viral regulatory elements and/or exogenous regulatory elements which for instance drive the expression of the exogeneous therapeutic gene product present in the vector. In otherwise successful gene therapy trials, these types of effects have resulted in otherwise unexplained cases of spontaneous leukemia and death in some of the patients.
These major secondary effects have been reported in gene therapy trials in patients with X-linked severe combined immunodeficiency (SCID-X1) with five reported cases of vector induced leukaemia found both in the Paris & London gene therapy trials. Similar effects have also been seen in several other gene therapy trials, for instance X-linked chronic granulomatous disease (X-CGD). These observed side effects of integrative vector mediated gene therapy, reveal current limitations of integrative vectors for gene transfer (Cavazzana-Calvo et al, 2000 and Hacein-Bey-Albina et al, 2003) formerly usedin clinical studies.
It has been shown that the integration of murine leukaemia virus (MLV) based vectors in the SCID-X1 patient was not random. Integration occurred mainly within or close to 5′ regulatory regions of transcriptionnally active genes. Insertional mutagenesis can result in acute toxicity (i.e. loss of transduced cells due to mutations of essential genes) or delayed side effects such as cancer induction. The nature and pathogenicity of these delayed side effects are highly context-dependent and will depend in part upon differences in the type of vector, transgene cassette, target cell, transduction conditions (copy number per cell) and disease-specific in vivo conditions for the maintenance and expansion of gene-modified cells.
The addition of genetic insulator elements (GIE) in integrating vectors which are used for gene therapy is an increasingly important avenue of research, in order to circumvent genotoxicity arising from insertional mutagenesis. Whilst the concept of gene therapy using integrating vectors was formulated some time ago and the idea of insulating the vector and genome from each other so as to prevent regulatory cross talk has also been known for sometime, suitable vectors have not yet been generated and therefore efficient and safe gene transfer vectors which would allow routine clinical use in human patients are still not readily available.
Most experiments/clinical trials performed so far with insulated retroviral vectors incorporate either the 1200 bp long HS4 insulator from chicken beta globin or a 250 bp long core sequence from this insulator as single or double copy cloned into the virus LTR (Ye et al., (2003)). It has now been shown that the 1200 bp HS4 insulator is not genetically stable in viral constructs; and it has also been established that the core sequence when present in one or two copies does not shield adjacent genomic neighbouring sequences against unwanted activation by the enhancer/promoter combination driving transgene expression.
There is thus a need to identify alternative sequences which both have enhancer-blocking activity and which have a boundary effect, as well as being stable in the virus and having no major effects upon virus biology and replication.
The present invention therefore relates to a new class of directly or indirectly integrating gene transfer vectors, which comprise in the nucleotide sequence to be integrated into the genome a GIE which prevents or significantly lessens the effects of the integrated sequences upon genomic sequences and vice versa the effects of genomic sequences upon the integrated sequence.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention there is provided a gene transfer vector which comprises at least one genetic insulator element (GIE), wherein said GIE comprises at least two copies of an element selected from the group consisting of:
a first CTCF binding site and a second CTCF binding site, wherein said first and said second CTCF binding sites are derived from the regulatory sequences of different genes;
a CTF binding site.
The inventors have developed a new class of integrating gene transfer vector comprising a new class of GIE which comprise multiple copies of either a CTCF or CTF binding site.
The inventors using different combinations of these elements have established several new species of GIE which they have shown to effectively insulate an exogenous gene encoded in an integrated gene transfer vector, following integration from genomic regulatory interference.
In particular the gene transfer vector (GTV) is an integrating gene transfer vector.
In the present Patent Application the term integrating gene transfer vector (IGTV), is intended to mean a molecule or collection of molecules which allow an exogenous sequence of DNA to be inserted into the genome of a recipient cell. In particular the IGTV may be based upon a virus such as a retrovirus or alternatively upon such materials as retroelements, transposons, liposomes and nanoparticles. In all cases the vectors comprise a nucleotide sequence which due to an innate or enzyme mediated mechanism is inserted into the genome of a host cell either randomly or alternatively at a specific locus (loci). All prior integrating gene transfer vectors share the genotoxic side effects known from examples of such vectors used in clinical trials.
Alternatively the gene targeting vector is a non-integrating gene transfer vector.
In the present Patent Application the term non-integrating gene transfer vector is intended to mean, a molecule or collection of molecules which allow an exogenous sequence of DNA to be expressed in the nucleus or cytoplasm of a transformed cell, but which are not integrated into the genome of the transformed cell. Suitable systems include episomal gene delivery systems, circular DNA molecules including minicircles, mini chromosomes and other suitable methods, including Integration Deficient LentiVectors (IDLV) platform. Although such non-integrating gene transfer vectors have the advantage that they are not expected to inherently have the genotoxic effects associated with IGTVs, a certain percentage of nuclei transformed with these non-integrating vectors are expected to integrate in any event due to random processes such as recombination and DNA repair. Non-integrating gene transfer vector comprising one or more GIE which would act to insulate the non-integrating gene transfer vector from the genome following such a random insertion and vice versa are also encompassed by the present invention therefore. At the present time it is estimated that anywhere up to 2% of non-integrating gene transfer vectors can end up genomically integrated. Therefore the shielding of the contents of these non-integrating gene transfer vectors is another important aspect of the present invention. Once such non-integrating gene transfer vectors, integrate into the genome they become IGTVs and therefore are explicitly comprised within the IGTVs according to this first aspect of the present invention.
In particular therefore the present invention relates to a non-integrating gene transfer vector comprising at least one genetic insulator element as defined above.
CCCTC-binding factor (CTCF) (“CCCTC” disclosed as SEQ ID NO: 31) is a well known regulatory protein whose function in various regulatory and developmental pathways continues to be elucidated (Ohlsson et al., (2001) and (Tae et al., (2007)) and whose consensus binding site is known (Tae et al., (2007)). The inventors have shown that by combining CTCF binding sites derived from the regulatory sequences of different genes, novel functional GIEs can unexpectedly be generated and importantly that when these GIEs are incorporated into an GTV that regulatory cross talk between vector encoded coding sequences and genomic enhancer/promoter elements is reduced.
In the present Patent Application the term derived from, is intended to mean a nucleotide sequence which is created from a parent nucleotide sequence and hence the derivative shares homology with the parent sequence, but may comprise one or more differences from the parent sequence. In particular the derivative nucleotide sequence may be a portion of the parent sequence or may comprise one or more nucleotide changes in comparison thereto.
According to a further aspect of the present invention the CTCF binding site in the GIE is characterised in that it contains a CTCF consensus binding site or has been experimentally shown to be bound by CTCF in vivo or in vitro. The CTCF binding site consensus (SEQ ID NO: 27; SEQ ID NO: 31) and methods to determine CTCF binding to a DNA target are provided in Tae et al., (2007) which is incorporated by reference.
Throughout this Patent Application references to particular sequences such as DNA binding sites and consensus versions of these, refer to both the provided forward sequence and to the complimentary reverse sequence.
According to a further aspect of the present invention the CTF binding site in the GIE is characterised in that it contains a CTF consensus binding site or contains a sequence which has been experimentally shown to bind CTF in vivo or in vitro. The CTF binding consensus and methods to determine CTF binding to a DNA target are provided in Tarapore et al., (1997) which is incorporated by reference.
CCAAT (SEQ ID NO: 30) box-binding transcription factor (CTF) or Nuclear factor I (NF-I) (also known as TGGCA-binding proteins) are a family of vertebrate nuclear proteins which recognize and bind, as dimers to specified DNA targets (Rupp et al., (1990)). The consensus binding sites of CTF (SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30) are known from Tarapore et al., (1997) and other sources which are hereby incorporated by reference.
In addition CTCF and CTF binding sites according to the present invention may share a certain level of homology or identity with the listed and other known consensus binding sites.
In particular by a homologous CTCF/CTF binding site it is intended to mean a sequence having at least 50% identity, preferably 60% identity and more preferably between 65 and 100% with one of the CTCF and CTF binding sites or consensus binding sites detailed in the present Patent Application or known in the art.
By identity it is intended to mean, a measurement of identity between two nucleic acid molecules. Identity can be determined by comparing a position in each sequence which may have earlier been aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings.
Each of the new GIEs the inventors have developed is smaller than the previously used 1200 bp insulator element and hence is expected to be more stable and to have less effect upon virus biology and infectivity. These new elements are also more functionally active than the previous insulators used as they comprise multiple copies of the essential core binding DNA sequences which provide the insulatory effect and less extraneous matter.
In particular the GIE is disposed in the GTV such that following genomic insertion of the GTV, the GIE is disposed between a genomic sequence and the coding sequences of the GTV.
In particular the first and/or second CTCF binding site or CTF binding site are derived from a CTCF binding site or CTF binding site from the regulatory sequence of a gene from an animal.
The inventors have combined CTCF binding sites from different species. The identification of alternative CTCF and CTF binding sites is possible given the sequence conservation of these sites (Xie X et al., (2007); Kim et al., (2007); Tae et al., (2007); Tarapore et al., (1997)) and such sequences are therefore encompassed by the present invention.
In particular the CTCF binding site is from the regulatory sequences of a gene selected from the group: betaglobin, immunoglobulin lambda locus, secretory protein LOC348174, similar to peripheral-type benzodiazepine receptor-associated protein 1, neuroblastoma breakpoint family, member 15, DKFZp434A0131 protein isoform 1, FKBP6-like, tripartite motif-containing 73, nuclear pore complex interacting protein, NODAL modulator 2, DKFZp434A0131 protein isoform 1, TBC1 domain family member 3C, FRMPD2-related 1, similar to cis-Golgi matrix protein GM130, nuclear pore complex interacting protein, TBC1 domain family member 3C, MUC1 mucin isoform 1 precursor, RD RNA-binding protein, T cell receptor betaneurotrimin, protocadherin gamma subfamily A, large conductance calcium-activated potassium, cell receptor alpha locus, p53-induced protein, zinc finger protein 384.
In particular the CTCF binding site is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 27; SEQ ID NO: 31.
The inventors have used two CTCF binding sites, CTCF1 which comes from regulatory sequences of chicken beta globin (SEQ ID NO: 7) and CTCF2 comes from the regulatory sequences of the human T cell receptor alpha/delta locus (SEQ ID NO: 8) and the CTCF binding sites therein have 64.3% and 78.6% identity with the CTCF consensus 1 (SEQ ID NO: 27) and 100% and 60% with CTCF consensus 2 (SEQ ID NO: 31) respectively.
HS4 is a DNase I-hypersensitive site isolated from the chicken beta-globin gene and described in Bell et al., 1997. Other examples of CTCF binding sites are given in Bell et al., 1997 and these are incorporated by reference into the present Patent Application.
In particular the GIE comprises the sequence SEQ ID NO: 6, which consists of the CTCF1 and CTCF2 sequences.
In particular the GIE comprises between three and six copies of the first and said second CTCF binding sites.
In particular the GIE comprises three copies of said first and said second CTCF binding sites.
In particular the GIE may comprise two or more identical CTF binding domains or alternatively the GIE may comprise two or more different CTF binding domains.
In particular the CTF binding site comprises the sequence SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30.
The CTF binding site used by the inventors (SEQ ID NO: 26) has 58% sequence identity with CTF Consensus 1 (SEQ ID NO: 28); 85% sequence identity with CTF consensus 2 (SEQ ID NO: 29) and 100% identity with CTF Consensus 3 (SEQ ID NO: 3).
The inventors have used a particular CTF binding site from the human oncogene Ski (Tarapore et al., (1997)) to validate the use of this binding site as a component of the GIE. In addition CTF binding sites from other genes may also be used and are encompassed by the present invention.
In particular the GIE comprises between two and eight copies of said CTF binding site.
In particular the GIE comprises four copies of the CTF binding site.
In particular the IGTV is a retrovirus vector.
Retrovirus vectors are based upon retroviruses, this group of viruses has a very characterisitic genomic structure comprising at either end of the linear DNA genome, (that is the genome produced by reverse transcription of the RNA genome), this comprises two LTR regions (also known as UTR) which each comprise a U3, R and U5 regions in that order. Contained between these LTR regions are the coding and regulatory sequences of the retrovirus and it is into this central portion of the retroviral genome that sequences encoding therapeutic gene products are inserted.
In particular the retrovirus vector is a gammaretrovirus or lentivirus vector.
Further examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, spumavirus (Coffin, (1996)).
In particular the GIE replaces at least a part of a U3 region of said retrovirus vector. Similarly the replacement can be made in another portion of the virus.
In retroviruses, the deletion of one or more essential elements such as the virus enhancer generates a disabled vector known as a self-inactivating construct (SIN) with reduced virus titer and infectious potential. Such constructs following genomic insertion can not generate further infectious virions. SIN vectors have been shown to be less prone to tumour-induction (Montini et al, 2006 & 2007) but do retain some of the oncogenic potential of unmodified vectors.
Alternatively, the retrovirus vector may be a SIN vector prior to GIE insertion.
In particular the retroviral vector comprises an enhancer.
According to the present invention an enhancer is a DNA sequence or a fragment thereof which when placed in functional combination with a sequence encoding a gene causes an increase in the expression of the gene.
The inventors when constructing their retroviral vectors deleted the endogenous viral enhancer when replacing the U3 portion of the retrovirus vector. They found that this reduced the expression level of a gene expressed by the retrovirus. The inventors have therefore modified the retroviral vector further so as to include an enhancer to increase gene expression levels.
In particular the exogenous enhancer is positioned between the GIE and an exogenous transgene encoded by the retrovirus vector.
In particular the exogenous enhancer is selected from the group consisting of viral enhancers, eukaryotic enhancers, animal enhancers, mammalian enhancers.
Genetic elements which can enhance the expression of adjacent genes are well known and all such enhancer elements are within the scope of the present Patent Application. The enhancer can be endogenous to the viral vector or to the virus from which the vector was derived or alternatively can be exogenous if for instance it comes from a eukaryotic source.
In particular the enhancer comprises the sequence SEQ ID NO: 22. The inventors have used a 133 bp long fragment extending from base 7708 to 7841 of the helper FB29 Fr-MuLV virus as the enhancer in the experiments described herein.
In particular the retrovirus vector comprises a polyA tail.
The inventors have shown that the presence of a polyA tail can significantly increase virus titers.
A polyA tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation. Numerous examples of polyA tails are known in the prior art and these are encompassed by the present invention.
In particular the polyA tail is located outside of the sequences derived from the retrovirus. Most particularly the polyA tail is located outside of the U3 of the retrovirus vector.
In particular the polyA tail comes from the human beta globin gene and comprises the sequence SEQ ID NO: 24.
In particular the retrovirus vector comprises a sequence selected from the group: SEQ ID NO: 12 to 19.
Alternatively the present invention also relates to a IGTV based upon a viral vector selected from the group, adenovirus, parvovirus (e.g. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpes virus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example (Coffin, (1996)).
In particular such non-retrovirus vectors comprise at least one GIE according to the first aspect of the present invention.
Alternatively the invention relates to an IGTV based upon a non-viral vector system such as naked DNA administration, minicircle, electroporation, liposomes, ultrasound, transposase-based and/or homologous recombination mediated insertion methods.
According to a second aspect of the present invention there is provided a cell genetically altered or modified using a retroviral vector according to the second aspect of the present invention.
A cell according to the present invention may be made according to a method, comprising at least the step of:
(a) introducing into a cell, a viral vector, as defined above and thereby generate a genetically modified cell having at least one integrated provirus copy of the viral vector, and
(b) isolating the genetically modified cell of step (a), by any appropriate mean.
The viral vector can be transfected into the cell or transduced.
The cell which is modified may be any cell of interest and stem cells in particular. For making transgenic/knock-out animals, the cells are pluripotent precursor cells such as embryo-derived stem (ES) cells, which are well-known in the art. For making recombinant cell lines, the cells may advantageously be human cells.
The present invention also relates to transgenic animals generated using viral vectors according to the present invention and to therapeutic and non therapeutic gene therapy methods using the viral vectors according to the present invention.
A further aspect of the present invention therefore is to identify short genetic elements that can be integrated into IGTV and in particular SIN (self-inactivating) vectors which prevent such unintended activation effects.
According to a third aspect of the present invention therefore, there is provided a method to identify GIEs suitable for use in a GTV involving the steps:
i) generating at least one first construct which comprises a transgene driven by a promoter; and at least one putative GIE, wherein said at least one putative GIE is disposed between said transgene and a genomic sequence following genomic integration of said at least one construct,
ii) generating a second construct comprising said transgene driven by said promoter,
iii) integrating at least one of said first and said second constructs into the genome of a eukaryotic cell,
iv) comparing the expression level of said transgene from said integrated first construct to the expression level of said transgene from said integrated second construct,
v) determining the presence of a GIE wherein said expression levels from step iv) are not identical.
In particular the first and second constructs may be IGTVs as described in the present Patent Application.
Alternatively the first and/or second constructs may be non-integrating gene.



BRIEF DESCRIPTION OF THE DRAWINGS
For a better understanding of the invention and to show how the same may be carried into effect, there will now be shown by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:
FIG. 1 shows a schematic representation of selected A. Gammaretrovirus vectors and B. Lentivirus vectors.
FIG. 2 shows the percentage of GFP producing TE-671 cells for various Gammaretrovirus constructs.
FIG. 3 shows the evolution A. of mean fluorescence in HeLa cells over time and B. percentage of HeLa cells fluorescing over 12 consecutive weeks in culture with gammaretroviral vectors.
FIGS. 4A-D show transduction of human TE 671 cells with various lentivirus vectors.
FIG. 5 shows the evolution A. of mean fluorescence in HeLa cells over time and B. percentage of fluorescing over 12 consecutive weeks in culture with lentiviral vectors.
FIG. 6A shows the genetic stability of the insulator 2 sequence provided as four repeats over 12 consecutive weeks of HeLa cells culture; 6B show the genetic stability of 8×Ins2 and 6×Ins1 in HeLa transduced with DCaro8.22 and DCaro6.14.
FIGS. 7 A-C show transduction of human CD34+ cells with various lentivirus vectors at 9 weeks
FIG. 8 shows the evolution A. of mean fluorescence in human CD34+ cells over 12 consecutive weeks in culture and B. percentage of human CD34+ cells fluorescing over time with various lentivirus vectors.
FIG. 9 shows the evolution A. of mean fluorescence in human CD34+ cells over 5 weeks and B. percentage of human CD34+ cells fluorescing over 5 weeks with various lentivirus vectors at MOI 5 and MOI 50.
FIG. 10 shows a restriction map of FrMuLV LTR.
FIG. 11 shows a restriction map of LTR FrMuLV Delta U3.
FIG. 12 shows a restriction map of Test 1 cPPT caro also named Lenp29.
FIG. 13 shows a schematic representation of the PCR cycling conditions for the amplication of different GIEs.
FIG. 14 shows a restriction map of Test 1 cPPT U3 eGFP 4×CTF also named DCaro4.23.
FIG. 15 shows a restriction map of pGEMT-FrUTR-Not-Eag.
FIG. 16 shows a restriction map of pGEMT-BetaGlobUTR.
FIG. 17 shows a restriction map of FOCHa-U3-eGFPsens-FrUTR-DeltaLTR6×CTCF.
FIG. 18 shows a restriction map of FOCHa-U3-eGFPsens-FrUTR-DeltaLTR6×CTCF-betaGlobUTR.
FIG. 19 shows a restriction map of pGEMT-hpGK-eGFP.
FIG. 20 shows a restriction map of FOCHa-hPGK-eGFPsens-FrUTR-DeltaLTR6×CTCF.
FIG. 21 shows an image of hES SA01 transduced with the DCaro4.23 insulated lentiviral vector on day 6 after transduction. Clear fluorescent transgene expression is seen in those human embryonic stem cells transduced with the vector.
FIG. 22A shows number of unique integration sites over the time course of the genotoxicity experiment for the pSIN18 (Ref1), Test1-cPPT/Lenp29 (Test1) and DCaro4 vectors; FIG. 22 B shows number of unique integration sites over the time course of the genotoxicity experiment for the pSIN18 (Ref1), Test1-cPPT/Lenp29 (Test 1) and DCaro4.23 vectors adjusted so that the total number of sequence sampled was 1000.
FIG. 23 shows a chart of the relationship between the vector copy number (VCN) and % GFP expressing cells over 12 weeks for HELA cells transduced with pSIN18 (Ref1), Test1-cPPT/Lenp29 (Test1) and DCaro4.23 vectors.
FIG. 24 shows the chromosomal distribution of integration sites in HELA cells transduced with pSIN18 (Ref1), Test 1-cPPT/Lenp29 (Test 1) and DCaro4.23 vectors.
FIG. 25 shows the distribution of integration sites in or close to genes, in HELA cells transduced with pSIN18 (Ref1), Test 1-cPPT/Lenp29 (Test 1) and DCaro4.23 vectors.
FIG. 26 shows the distribution of integration sites in genes or gene regulatory sequences, in HELA cells transduced with pSIN18 (Ref1), Test1-cPPT/Lenp29 (Test1) and DCaro4.23 vectors.



DETAILED DESCRIPTION
There will now be described by way of example a specific mode contemplated by the Inventors. In the following description numerous specific details are set forth in order to provide a thorough understanding. It will be apparent however, to one skilled in the art, that the present invention may be practiced without limitation to these specific details. In other instances, well known methods and structures have not been described so as not to unnecessarily obscure the description.
Example 1
Materials and Methods
1.1 Generation of Enhancer-Deleted retrovirus LTRs: Self Inactivating Deletions (SIN)
a. Gammaretroviruses
From the above native FrMuLV LTR (FIG. 6, SEQ ID NO: 1), a SIN-24 ΔU3LTR version was derived through the deletion of a 388 bp long sequence, from the U3 sequence, resulting in LTR FrMuLV Delta U 3 (FIG. 7, SEQ ID NO: 2).
In a further step, a BamH1-ClaI polylinker (SEQ ID NO: 3) was added in between base 24 and the start of the R portion of the vector, in order to be able to clone the insulator sequences in later experiments. This step was performed using PCR and the resulting modified LTR sequence was verified (SEQ ID NO: 4).
b. Lentiviruses
pSIN-18
pSIN-18 3′ LTR vectors (SEQ ID NO: 5) were used as established by Zufferey et al., (1998). A synthetic 118 bp cPPT sequence encompassing a 5′ HpaII and a 3′ClaI sites was inserted in the original vector natural ClaI site (position 4010).
LENP29
LENP29 derives from pSIN-18 PGK from which the hPGK-eGFP-WPRE fragment (EcoRV-KpnI) has been removed and replaced by a polylinker (EcoRV-EspI-MluI-BamHI-SalI-KpnI). The U3 region of Friend 29 was obtained by EspI-BssHI partial digestion of FOCHA-FICD and cloned in the compatible sites of the vector polylinker (EspI-MluI). The EGFP1 cDNA was cloned downstream of this internal promoter element. The 18 remaining bases of the U3, in the pSIN-18 3′LTR vector include a BbsI site which was used for the cloning of each insulator element as a BbsI fragment, the non-symetrical ends generated by BbsI digestion allowed the correct orientation of the insulator fragment in the vector.
1.2 Sequences of the Insulators
Insulator1: CTCF 1 CTCF2 repeated 3 times called: “6×CTCF”
This insulator was synthetised as overlapping complementary single stranded oligonucleotides, which were sequentially hybridised to form the multimerised 6×CTCF (SEQ ID NO: 6), which comprises three repeats of the CTCF1 FII site (SEQ ID NO: 7) was derived from the HS4 chicken beta-globin insulator and CTCF2 (SEQ ID NO: 8) was derived from the human T cell receptor alpha/delta locus BEAD-A. 6×CTCF comprises a 5′ BamHI site and a 3′ ClaI site.
Insulator 2: Multimerised CTF
Multimerised CTF sites consist of various numbers of repeats of DNA sequences containing CAAT transcription factor (CTF) binding sites which were isolated from pNF7CAT plasmid (Tarapore P et al NAR 25: 3895-903, 1997) and analysed by DNA sequencing. Flanking sequences contain appropriate restriction enzyme cleavage sites, 5′ ClaI-HindIII and 3′ BamHI. Three versions Multimerised CTF sequences were generated 2×CTF Insulator (SEQ ID NO: 9); 4×CTF Insulator (SEQ ID NO: 10) and 7×CTF Insulator (SEQ ID NO: 11). These sequences are provided for the negative strand and following cloning into the relevant vector read along the positive strand.
1.3 Cloning of the Insulators into the SIN LTRs
Gammaretrovirus
The selected insulator fragment and LTR FrMuLV Delta U 3 were each digested with BamHI and ClaI as per the supplier instructions. The digested insulator fragments and vector were then ligated and correctly ligated fragments were verified by sequencing selected clones. The sequence of the modified vectors are LTR FrMuLV Delta U 3 6×CTCF (SEQ ID NO: 12); LTR FrMuLV Delta U 3 2×CTF (SEQ ID NO: 13); LTR FrMuLV Delta U 3 4×CTF (SEQ ID NO: 14); LTR FrMuLV Delta U 3 7×CTF (SEQ ID NO: 15).
Lentivirus
Insulator fragments were cloned into the Lenp29 vector following digestion of the vector with BbsI and the insulator nucleotide molecules with BamHI and ClaI. The sequences of the modified vectors are Lenp29 6×CTCF (SEQ ID NO: 16); Lenp29 2×CTF (SEQ ID NO: 17); Lenp29 4×CTF (SEQ ID NO: 18); Lenp29 7×CTF (SEQ ID NO: 19). The digested insulator fragments and vector were then ligated and correctly ligated fragments were verified by sequencing selected clones.
The cloning strategy to generate DCaro4.23 (SEQ ID NO: 42) was as follows, Test1-cPPT/Lenp29 (FIG. 8, SEQ ID NO: 34) was digested as per manufacturers instructions with BbsI. Test1-cPPT was linearised by this digestion as the plasmid comprises a unique BbsI site. Digested plasmids were run upon an agarose gel and linearised fragments were recovered by gel purification.
Each insulator was amplified by PCR using Taq Phusion, in order to insert a BbsI site at both ends.
PCR was performed using the following primers for the amplification of the 2×CTF, 4×CTF and 7×CTF using the following primers:









* Sense oligo: (Ins_BbsI_472sens)



(SEQ ID NO: 20)



       BbsI         ClaI



 



5′- AAGAAGACAAGATCATCGATAAGCTTGCATTGGC -3′



 



* Reverse oligo: (lns_Bbsl_4-7rev)



(SEQ ID NO: 21)



              BbsI BamHI



 



5′- TTTGATCAAGTCTTCGGATCCCCCAATTCGC -3′






PCR was performed using the following primers for the amplification of the 6×CTCF element:









* Oligo sens: (Ins_BbsI_6sens)



(SEQ ID NO : 32)



5′- AAGAAGACAAGATC_GGATCCCCCAGGGATGTAAT -3′



 



* Oligo reverse: (Ins_BbsI_6rev)



(SEQ ID NO: 33)



5′- TTTGATCAAGTCTTC_ATCGATTGGAGCTCCCCGG -3′






Amplified fragments were then digested with BbsI and purified digested fragments were ligated into the previously BbsI digested plasmid Test1-cPPT, recircularising the plasmid when successfully inserted and generating e.g. the DCaro4.23-eGFP or Test1-cPPT-U3-eGFP-4×CTF plasmid (FIG. 10).
1.4 Fr-MuLV 3′UTR Fragment
The sequence of a 133 bp long fragment extending from base 7708 to 7841 of the helper FB29 Fr-MuLV virus (Cohen-Haguenauer et al., (1998)) is as follows:
TAGTTCAATTTGTTAAAGACAGGATCTCAGTAGTCCAGGCTTTAG TCCTGACTCAACAATACCACCAGCTAAAACCACTAGAATACGAGCCACAATAAAT AAAAGATTTTATTTAGTTTCCAGAAAAAGGGGGG (SEQ ID NO: 22), this sequence includes an extra stretch of bps which comes from the cloning strategy and does not affect the performance of this element.
This fragment was generated by PCR and cloned as an intermediate in a pGEMT shuttle (Promega) generating pGEMT-FrUTR. The Fr-MuLV 3′ UTR was cloned by ligating a NotI-EagI fragment from the pGEMT-FrUTR (FIG. 11) to each recipient vector digested at their NotI site.
The sequence of the 3′ end of the insulated gammaretrovirus vector named pFOCHa-(U3-eGFP)sens-Fr3′UTR-ΔU3-7-CTF (p23 see Table 1 below), is provided as SEQ ID NO: 23.
1.5 Synthesis of the Beta-Globin polyA Fragment
A synthetic sequence has been designed and synthetised as follows:








(SEQ ID NO: 24)



gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatc



 


tggattctgcctaataaaaaacatttatttttcattgca.






This sequence was inserted into a pGEMT shuttle (Promega), generating the pGEMT-betaGlobpolyA shuttle plasmid, from which it can be retrieved as a 161 bp long BglII fragment (SEQ ID NO: 24) and further oriented using an internal XhoI site (FIG. 12).
As an example of this cloning regime, the 161 bp BglI fragment from pGEMT-betapolyA was cloned into p11 (FOCHa-U3-eGFPsens-3′UTR Fr-ΔU3 6×CTCF (FIG. 13)) so as to form p14 (FOCHa-U3-eGFPsens-3′UTR Fr-ΔU3 6×CTCF-betaGlobpolyA (FIG. 14)).
Following all cloning experiments the selected constructs were sequenced to determine the correct orientation and sequence of the constructs.
1.6 Internal Promoters
Each internal promoter used comprised sequences as follows:








Fr-MuLV U3



(SEQ ID NO: 25)



Ctcgagaagcttgatatcgcnagcctgatagccgcagtaacgccattagcaaggcatggaaaaataccaa



 


accaagaatagagaagttcagatcaagggcgggtacacgaaaacagctaacgttgggccaaacaggatatctgcggtgagcagtttc


 


ggccccggcccggggccaagaacagatggtcaccgcggttcggccccggcccggggccaagaacagatggtccccagatatggc


 


ccaaccctcagcagtttcttaagacccatcagatgtttccaggctcccccaaggacctgaaatgaccctgtgccttatttgaattaaccaat


 


cagcctgcttctcgcttctgttcgcgcgcttctgcttcccgagctctataaaagagctcacaacccctcactcggcgcgtcgcggaattcc


 


gcggatcc.






This sequence comprises a XhoI and HindIII restriction site at the 5′ end (underlined) and an EcoRI and BamHI restriction site at the 3′ end (underlined).
hPGK
The initially used ‘high expression level’ internal promoter (Fr-MuLv U3) was replaced by a housekeeping ‘low expression level’ hPGK promoter, in each of the insulated constructs giving rise to accurate virus titres, in order to test for both virus production and transgene expression together with genotoxicity characteristics according to the internal promoter driving the expression of the insulated vectors. Each construct is named by adding hP to its parental U3 counterpart (see Table 1, first column).
The hPGK promoter was in combination with the eGFP coding sequence was excised from the pSIN-18 vectors as described above.
The hPGK-eGFP sequence can be excised from its pGEMT-hPGK-eGFP (FIG. 15) shuttle by XhoI digestion and in turn replaces the U3-eGFP XhoI fragment in the recipient insulated vector construct.
An example of the replacement of the U3-eGFP fragment, FOCHa-(U3-eGFP) sens-3′UTR Fr-ΔU3 6×CTCF (FIG. 13) was digested with XhoI as per suppliers instructions, pGEMT-hPGK-eGFP was similarly digested with XhoI and fragments of the correct size were isolated and purified using agarose gel separation and isolation.
Purified FOCHa-(U3-eGFP) sens-3′UTR Fr-ΔU3 6×CTCF and pGEMT-hpGK-eGFP fragments were then ligated and recircularised plasmids were obtained and sequenced to ensure they contained the correct insert and were the final plasmid FOCHa-(hPGK-eGFP) sens-3′UTR Fr-ΔU3 6×CTCF (FIG. 16).
The replacement of the Fr-MuLV U3 enhancer-promoter by the hPGK promoter was performed in both gammaretrovirus constructs and lentivector constructs. For example data obtained with Dcaro4hP are presented on cord blood derived Human Stem Cells shown in FIGS. 7, 8 and 9.
1.7 Transfections, Virus Production and Transduction Procedures Retrovirus-Producer Clones and Primary Evaluation of Transduction Capacity
The third generation of TE-FLY-GA GALV producer cell-lines (kindly forwarded by F-L Cosset) have been engineered with each gamma vectors via primary transfection using co-transfection with a G418 resistance plasmid using SuperFect (Qiagen). Neomycin-resistant clones were pooled and stable producer cells were grown before and after selection of cells expressing EGFP1 by cell-sorting. A helper-virus mobilisation assay was developed using two index-cell lines which allow the detection of GALV pseudotypes (detection of either GFP positive or G418-resistant cells following helper-mobilisation). No helper virus could be detected in any of the tests performed over years, on virus batches produced.
For the lentivectors, quadruple transfections on 293T cells and vector supernatant harvest (including concentration of virus particles using ultracentrifugation) were performed as previously described (Dull et al., (1998). Titers were on average in the 109 to 1010 cfu/ml range, depending on the vector construct produced and based on both p24 and PCR assays and functional infectious titers from GFP positive cells measured by FACS, at dilution E-5, E-6, E-7 in order to prevent false positive data when further dilution ranges are used
Cell Cultures
Virus producing cells, 293-T, TE-671 cells and HeLa cells (used for titers/assays) were grown in DMEM with 10% FCS serum, 2 mM of L-Glutamine and 50 μg/ml of gentamycine.
Hematopoietic Cells Transduction.
Cells were thawed in SCGM with 1 mM NAC and 100 ng/ml TPO, and seeded ON in 12 wells plates. Anti-TGFβ (10 μg/ml) was added approx 16 hours before transduction. One cycle of infection was applied the next day at MOI 5-10 in SCGM medium containing: NAC (1 mM), TPO (25 ng/ml), LIF (25 U/ml), SCF/MGDF (25 ng/ml) and Flt-3 (25 ng/ml). After transduction, cells were placed in long-term culture (LTC).
Long Term Culture Conditions
Cells were seeded in liquid LTC culture in SCGM under low concentrations of growth factors: TPO (25 ng/ml), LIF (25 U/ml), SCF/MGDF (25 ng/ml), Flt-3 (25 ng/ml), for the first 6 weeks; from week 7 on, the culture medium included: SCGM medium with TPO (5 ng/ml), LIF (10 U/ml), SCF/MGDF (25 ng/ml), Flt-3 (5 ng/ml), IL3 (5 U/ml), G-CSF (10 U/ml) and Epo (0.5 U/ml). Half of the supernatant was replaced each week. Weekly analysis consisted on counts, FACS analysis of GFP expression, CFUs & PCR assays.
Measurement of eGFP Expression/FACS
Cells were washed and analysed by FACS analysis according to the manufacturers standard protocol and settings (BD FACSCalibur System, Becton, Dickinson and Company) without further labelling. Depending on the samples under test, either 5000 to 50 000 cells are analysed. Sorting of GFP positive cells was performed on a cell-sorter (Vantage, Becton, Dickinson and Company) using standard settings and protocols.
Example 2
Results
2.0 Introduction
The inventors sought to design synthetic GIEs consisting of multimers of specified core sequences. The first one GIE (Insulator 1) is a new combination of CTCF binding sites: 42 bp FII sequence (CTCF1) derived from the HS4 portion of the beta globin gene and a 39 bp BEAD-A sequence (CTCF2) derived from the human T cell receptor alpha/delta locus. Insulator 1 consists of three repeats of the CTCF1-CTCF2 sequence. The second insulator (Insulator 2) comprises multimerised CTF sites which consist of various numbers of repeats of DNA sequences containing CTF binding sites which were isolated from the pNF7CAT plasmid (Tarapore P et al NAR 25: 3895-903, 1997).
A series of insulated gamma- and lenti-, SIN-retrovirus constructs have been engineered using several combinations of the shortest active stretches for the insulators tested, respectively of 268 bp for Insulator 1 and various number of repeats for Insulator 2 of either 181 bp (8×CTF), 157 bp (7×CTF), 101 bp (4×CTF) or 56 bp (2×CTF) long, substituting the U3 LTR enhancer in full in the recipient constructs.
2.1 Key Factors which are Necessary for the Production of Insulated Self-Inactivating Gammaretroviral Vectors with High Titres
All constructs designed were tested in comparison with two reference controls: the first, p7 (see Table 1 below) with native LTRs sequences and a strong internal promoter (positive control) and the second p1 a self-inactivating (SIN) vector with a full enhancer-deletion (negative control). A series of insulated vectors have been generated from the SIN-construct and tested, selected examples of these are shown schematically in FIG. 1 and all are listed in Table 1 below.







TABLE 1





List of gammaretrovectors constructed and tested







VECTORS with strong Fr-MuLV U3-promotor








Name
Structure of the construct





P1
FOCHa-(U3-eGFP)sens-ΔU3 (SEQ ID NO: 35)


 


p2
FOCHa-(U3-eGFP-polyA hβglob)antisens-AU3


 


P3
FOCHa-(U3-eGFP-polyA hβglob)antisens-AU3-6xCTCF


 


p4
FOCHa-(U3-eGFP)sens-ΔU3-6xCTCF


 


P5
FOCHa-(U3-eGFP-polyA hβglob)sens Fr3′UTR


 


p6
FOCHa-(U3-eGFP-polyA hβglob)antisens Fr3′UTR


 


P7
FOCHa-(U3-eGFP)sens-Fr3′UTR (SEQ ID NO: 36)


 


P8
FOCHa-(U3-eGFP)antisens-Fr3′UTR


 


P9
FOCHa-(U3-eGFP)sens-ΔU3-6xCTCF- polyA hβglob


 


p10
FOCHa-(U3-eGFP-polyA hβglob)antisens-ΔU3-6xCTCF-polyA hβglob


 


pll
FOCHa-(U3-eGFP)sens-Fr3′UTR-AU3-6xCTCF (SEQ ID NO: 37)


 


p12
FOCHa-(U3-eGFP-polyA h(3glob)antisens-Fr3′UTR-ΔU3-6xCTCF-polyA hβglob


 


p13
FOCHa-(U3-eGFP-polyA h(3glob)antisens-Fr3′UTR-ΔU3-6xCTCF


 


p14
FOCHa-(U3-eGFP)sens-Fr3′UTR-AU3-6xCTCF-polyAhβglob


 


p15
FOCHa-(U3-eGFP)sens-AU3-4xCTF


 


p18
FOCHa-(U3-eGFP)sens-AU3-4xCTF-polyA hβglob


 


p19
FOCHa-(U3-eGFP)sens-Fr3′UTR-ΔU3-4xCTF (SEQ ID NO: 38)


 


p20
FOCHa-(U3-eGFP)sens-Fr3′UTR-ΔU3-4xCTF-polyA hβglob (SEQ ID NO: 39)


 


p21
FOCHa-(U3-eGFP)sens-Fr3′UTR-ΔU3-2xCTF


 


p22
FOCHa-(U3-eGFP)sens-Fr3′UTR-ΔU3-2xCTF-polyA hβglob


 


p23
pFOCHa-(U3-eGFP)sens-Fr3′UTR-ΔU3-7xCTF


 


p24
FOCHa-(U3-eGFP)sens-Fr3′UTR-ΔU3-7xCTF-polyA hβglob










VECTORS with housekeeping hPGK-promoter








Name
Structure of the construct





p1hP
FOCHa-(hPGK-eGFP)-sens-ΔU3


 


p11hP
FOCHa-(hPGK-eGFP)-sens-Fr 3′UTR-DU3-6xCTCF(SEQ ID NO : 40)


 


p14hP
FOCHa-(hPGK-eGFP)sens-Fr3′UTR-AΔU3-6xCTCF-polyA hβglob


 


pl5hP
FOCHa-(hPGK-eGFP)sens-AΔU3-4xCTF


 


pl8hP
FOCHa-(hPGK-eGFP)sens-AΔU3-4xCTF-polyA hβglob


 


p19hP
FOCHa-(hPGK-eGFP)sens-Fr3′UTR-ΔU3-4xCTF


 


p2OhP
FOCHa-(hPGK-eGFP)sens-Fr3′UTR-ΔU3-4xCTF-polyA hβglob (SEQ ID NO: 41)


 


p21hP
FOCHa-(hPGK-eGFP)sens-Fr3′UTR-ΔU3-2xCTF-


 


p22hP
FOCHa-(hPGK-eGFP)sens-Fr3′UTR-ΔU3-2xCTF-polyA hβglob


 


p23hP
FOCHa-(hPGK-eGFP)sens-Fr3′UTR-ΔU3-7xCTF


 


p24hP
FOCHa-(hPGK-eGFP)sens-Fr3′UTR-ΔU3-7xCTF-polyA hβglob









The simplest constructs were tested first, these only included a splice donor and a splice acceptor followed by an internal promoter driving reporter gene expression, placed either in a sense or an antisense orientation, with reference to the direction of viral transcription.
The internal promoter initially used is a strong enhancer/promoter combination derived from the Fr-MuLV LTR, named “U3” hereafter, which drives transgene expression from the SIN-Insulated vectors in transduced cells. A weaker promoter from the human PGK gene was also used in later experiments.
The inventors decided to use eGFP1 as a reporter gene, the expression level of which was serially measured in both virus-producing and transduced cells, which were sustained in culture for over one month. Virus titers were evaluated from two distinct sources of virus-producer cells, either pools of virus-producer cells or individual clones derived from single-sorted cells seeded in limiting dilutions from those exhibiting the brightest levels of EGFP1 expression.
None of the tested constructs proved useful, as they failed to produce virus at a sufficient titer and at least one to two orders of magnitude less than the control p7 and p1 vectors (Table 1). None of the antisense constructs was able to generate detectable EGFP1 expression in test-target cells.
2.2 Adding the 3′UTR Sequence from the Fr-MuLV FB29 virus strain
The inventors then searched for additional features which would be capable of increasing insulated vectors titers, two rationales were followed and two distinct types of genetic elements were added.
3′UTR Sequence from the Fr-MuLV FB29
The first element is a 179 bp long 3′UTR sequence from the Fr-MuLV FB29 virus strain, which has been shown to play a critical role in the helper virus biology (Sitbon et al., 1986). Its addition in a downstream position located in between the transgene of interest and the 3′ LTR, results in a significant increase in virus titers, by one or two orders of magnitude, according to the insulator used. The most dramatic effect is observed with the Insulator 2 (4×CTF) series of constructs p19/p20, with an increase of virus titers to levels above the p7 control (FIG. 2). This improvement was seen only with constructs in the sense orientation, as antisense constructs did not appear to produce more detectable transduction units. Therefore, no antisense construct was considered in further steps.
PolyA Signal from the Human-β Globin Gene.
A second element was added which does not affect the sequences integrating into the host cell genome, the equivalent of the integrated provirus. The inventors designed and custom-synthetised a polyA sequence derived from the human-β globin gene which was placed outside the virus cassette, downstream of the 3′ LTR, in a position which operates in the virus producing cells only. The addition of this sequence downstream to the GIE is intended to facilitate the production of virus read through, with the assumption that the GIE could cause premature interruption of virus RNA synthesis. In fact, the addition of this sequence, was able to induce a dramatic improvement of virus titers with Insulator 2 containing constructs whereas the opposite effect was seen with Insulator 1, in the most dramatic way since virus production dropped to almost undetectable levels with p14 as compared to p11 (Table 2 and data not shown).
In a number of retroviral vectors, the woodchuck hepatitis post-transcriptional regulatory element (WPRE) has been used as a post-transcriptional stabiliser of virus-RNA. This sequence has been reported as potentially oncogenic (Kingsman et al, 2005). Therefore, it was not been placed in any of the retrovirus constructs (whether gammaretrovectors or lenti vectors) tested.







TABLE 2







Relative transduction efficiency of test TE-671 cells using raw virus


supernatant (i.e.: without applying selection towards isolation of best


virus producing cells).













Mean



Vector
% GFP+ cells
Fluorescence











U3 internal promoter driven expression











p1
0.87%
90.45



P7
0.75%
24.77



p11
0.40%
20.99



p14
0.06%
16.82



p19
5.05%
60



p20
18.81%
53.62







hPGK internal promoter driven expression











p7hP
2.52%
21.93



p11hP
0.24%
27.48



p14hP
0.06%
11.81



p19hP
8.49%
20.47



p20hP
12.78%
34.31










2.3 Comparing Insulator 1 and Insulator 2 and No Insulator in Retroviral Vectors
Transgene Expression Level
In general the lowest level of eGFP expression is recorded in constructs comprising, Insulator 1, as compared to reference LTR and SIN vectors and constructs comprising Insulator 2 with the same internal promoter, including the strong Fr-MuLV U3 sequence, suggesting a potential inhibitory or silencing effect of Insulator 1. With Insulator 2, a shift in the intensity of the GFP expression is recorded, with a dramatic increase of the mean GFP level and a more homogenous distribution of GFP intensity from one cell to the next, as observed on the FACS profiles. This holds true with both human target cell lines tested, i.e., TE-671 and HeLa with gammaretrovectors (Table 3) and both HeLa cells and human haematopoietic primary stem cells from cord blood followed-up during 12 consecutive weeks after transduction with various lentivector constructs (FIGS. 5 and 8 respectively; in FIG. 8 data shown with Insulator 2 only).







TABLE 3







HeLa test cells transduced with native, SIN & Insulated


GammaRVV and lentivectors












Producers cells






derived from

% of GFP+
Mean



vectors #

HeLa
Fluorescence
















p1
Sin
88
139



p7
Native-LTR
92
93



p11
Fr-U3EP Ins1
87
58



p15*
Fr-U3EP Ins2
89
213



p18*
Fr-U3EP Ins2
83
151



p11hP
hPGK-Pro Ins1
94
22







*= p15 & p18 are identical in transduced cells; they only differ in virus producing cells (polyA downstream the vector 3′LTR)






When lentivectors were tested, the same pattern of expression profile was observed with the lowest eGFP expression levels recorded in DCaro6.14 (Insulator 1) as compared to DCaro4.23 (Insulator 2) or reference vectors. In this case, the mean eGFP fluorescence intensity is brighter with the insulator 2 carrying vector as compared to pSIN-18 as shown in FIGS. 4, 5, 6 and 7 and Table 6.
Transgene Expression Stability
eGFP positive transduced HeLa cells were sorted 5 days after exposure to the vectors and sequentially passed ( 1/10th) each week and followed over a three months period.
With gammaretrovectors, the mean eGFP fluorescence intensity remained stable over time with all vectors tested, which ever insulator was used (FIG. 3A) but the percentage of eGFP expressing cells gradually decreased when transduced with Insulator 1 carrying vectors as shown in FIG. 3B, suggesting either a gradual silencing effect over time or genetic instability of insulator 1 placed in that context.
Again, FIG. 5B shows the dramatic decrease from 70 to 25% in the percentage of eGFP expressing cells with the lentivector including Insulator 1 (Dcaro6.14), over our 12 weeks follow-up in Hela cells. The same holds true in human haematopoietic primary stem cells from cord blood.
FIGS. 5A and 5B, clearly illustrate that the single vector which mediates stable and sustained expression of the transgene over time is Dcaro4.23, which includes four repeats of Insulator 2. With the two reference constructs, pSIN-18/Ref1 and Lenp29/Test1, the % of eGFP expressing cells dramatically decreased from 65% to 22% and 80% to 40% respectively while Dcaro4.23 remains in the 80% range. Similar observations can be made on the expression level which remains in the 600-700 mean eGFP fluorescence with Dcaro4.23, while it decreases with Ref1 and dramatically increases with Test1.
4× Insulator 2 Genetic Stability in Hela Transduced Cells
PCR was performed from primers in flanking regions of the insulators at both ends, leading to an 219 bp long amplicon in the presence of 4× Insulator 2 and to a 99 bp long amplicon with the non-insulated vector, e.g.: Test1/Lenp29. Where insulator 2 has been deleted or rearranged, it is expected that additional bands would have appeared in between 219 and 99 bp and in between week 2 to 11, which is not seen (FIG. 6A). Instability levels were also monitored for both DCaro8.22, which comprises 8 repeats of insulator2 and DCaro6.14, based on 6×CTCF (FIG. 6B).
2.4 Virus Titers
Similarly to transgene expression level, the addition of Insulator 2 resulted in much higher titres as compared to Insulator 1.
With gammaretrovirus constructs, the variation in titer between Insulator 2 and Insulator 1 viruses encompassed two orders of magnitude when comparing both identical Virus//different Insulator combinations directly. Interestingly, titers of both p19 and 20 were significantly higher than with the reference LTR-p7 control as shown in FIG. 2. With lentivirus constructs, titres of Insulator 1 and Insulator 2 carrying vectors also significantly differed, by an order of magnitude, as shown on Table 4 (p24 titres shown).











TABLE 4







Functional titre:
p24 titre
mean


Lentivector
Insulator
293 cells
pfu/ml
Fluorescence



















Lenp29/Test 1
N/A
9.75E+08
4.39E+09
951


pSIN-18/Ref1
N/A
2.09E+09
1.24E+10
257


DCaro4.23
4xIns2
1.17E+09
1.10E+10
667


DCaro6.14
6xIns1
3.55E+08
6.34E+09
47









2.5 Comparing Various Numbers of Insulator 2 Repeats in Retroviral Vectors
In order to investigate whether the number of CTF repeats present in Insulator 2 constructs impacts or not on the performances of these various constructs, both gammaretrovirus and lentivirus vectors were designed with either 2×CTF, 4×CTF or 7⁄8 CTF repeats as GIEs cloned into the deleted SIN LTRs.
The most interesting data were monitored with vectors containing the 4×CTF GIE (p20, Table 5A and 4× C1.1 Table 6B), wherein for both types of vector, mean fluorescence exceeded all other tested vector types. This preference holds true in reference both transgene expression levels and virus titres generated (Table 4), wherein for example the titer of p20 is on the 10E5 pfu/ml range but this is without the normal step of selecting the best producers; this is bulk producer cell pool and could translate easily into over 10E6 with additional selection procedures.
Surprisingly, 7⁄8×CTF (p24 in Table 6A and 7×c12 in Table 6B) did not perform better than 2× (p22 in Table 5A and 2× c16 in Table 5B) and in the case of p24 mean fluorescence was less than 50% of p22.
The inventors have also found some unexpected effects with the 7×CTF element which may be due to instability of this element in the virus, (or other as yet unexplained reasons). These effects were also seen during the preparation of plasmids comprising this element, for transfection.







TABLE 5A







Comparing various repeats of Insulator 2 in Gammaretrovectors












TF25




Plasmid
% GFP+
Mean



Name
cells
Fluorescence















p7
2.67%
24.77



p20
14.14%
48.85



p22
10.41%
24.04



p24
0.37%
19.70

















TABLE 5B







Comparing various repeats of Insulator 2 in Lentivectors












Number of
Mean



Vector
GFP+ cells
Fluorescence















4x cl.1
368
464.64



6x cl.8
143
136.84



2x cl.6
103
96.18



7x cl.2
166
81.42










Observations made with lentivectors corroborate these data to an even broader extent as shown on FIGS. 5A and 5B, where the multiplicity of infection (MOI) has been normalised so that the number of infectious virus particles is identical whichever vector is tested, irrespective of the initial difference observed in virus production titers. Therefore, using the same MOI, striking differences are monitored between the 4× Ins2 (Dcaro4.23) and the 8× Ins2 (Dcaro8.22) vectors: whether in the initial % of transduced cells with over 84% and 45% respectively; or during follow-up, after 12 weeks, Dcaro4.23 remains in the 80% range while Dcaro8.22 levels have dropped to 22%, half of the initial % of transduced cells. Mean fluorescence levels with the 8× Ins2 vector also are half of that observed with the 4× Ins2. Similar data have been observed in haematopoietic cells from cord blood (data not shown).
2.6 Use of Alternative Internal Promoters in Insulator 1 and Insulator 2 Gammaretroviral Vectors
Two different internal promoters, driving the expression of the transgene from the SIN/Insulated vectors were tested. The first one is a strong enhancer/promoter derived from the Fr-MuLV FB29 strain virus LTR, referred to hereafter as “U3”. Its characteristics have already been described (Sitbon et al, 1994; Cohen-Haguenauer et al, 1998 & 2006) and in particular its potential to express transgenes at high levels in both haematopoietic and CNS stem cells.
The second promoter used is the “hPGK” originating from a housekeeping gene, identified as a ubiquitous, low level sequence which is not susceptible to extensive variations expression level.
The two promoters have been selected according to prior observations and publications, which clearly show that the expression levels driven from the virus-derived promoter and the housekeeping promoter are distinct, although other factors such as the cell type may be a factor due to virus transcriptional tropism, while the housekeeping promoter will provide equal levels whatever the host cell.
While the intensity of expression was completely shielded and overall quite low and similar with Insulator 1 derived vectors whatever the strength of the internal promoter used, significant differences were monitored with vectors containing Insulator 2, as shown on FIGS. 3A, 5A, 7 and 8A. In heamatopoietic stem cells cultured over 12 weeks, the mean flurorescence differed by roughly a log range and remained stable as such over time. This observation demonstrates that Insulator 2 vectors mediate expression according to the strength of the internal promoter. The expression cassette is thus becoming strictly insulated and independent from neighbouring sequences and silencing.
In experiments using the control non-insulated vectors, as was expected lower expression levels were observed on average when the hPGK promoter was driving eGFP expression in comparison to when U3 was driving eGFP expression; an observation which was also seen with lentivectors as shown in Table 4, since pSIN-18 comprises a hPGK promoter (mean recorded eGFP fluorescence: 76) while Lenp29 comprises a U3 promoter (mean eGFP fluorescence: 144).
2.7 Transduction of CD34+ Cells from Human Cord Blood
Insulated Self-Inactivating lentiviral vector DCaro.23 has a high titer and mediates efficient transduction and sustained long-term expression into human cord blood haematopoietic stem cells, as shown in FIG. 7 and FIGS. 8A and 8B, with a 12 weeks follow-up. It is noticeable that both the expression level and the percentage of transgene expressing cells remain stable over time with Dcaro4.23 and DCaro4hP.
In addition it is noticeable that in the 4× Ins2 vectors, the mean expression level is conserved whatever the MOI under use, as shown in FIG. 9; while with Dcaro4.23 this level is sustained in the 700-800 range, over a prolonged period (FIG. 8A), with both Ref1 and Test1 there is a markedly distinct level of eGFP expression with a MOI of 50 is applied as compared to a MOI of 5. This striking observation reveals the remarkable power of the 4× Insulator 2 placed in the retroviral LTR context, since each trangene-expressing cassette integrant has become independent from its neighbouring genetic environment and a homogenous expression pattern is observed, which depends on the strength of the internal promoter placed between the two flanking insulator sequences.
Interestingly, the difference in the mean transgene expression level based on eGFP reporter is markedly increased in human primary haematopoietic stem cells as compared to other cell-lines, as shown on Table 6 and FIG. 8A, since as mentioned, a log-range expression level difference is observed with Dcaro4.23 (Fr-MuLV U3 as internal promoter) and DCaro4hP (with hPGK as internal promoter).












TABLE 6









LSIN-U3/Test1
4xCTF-U3/DCaro4.23
pSIN-18/Ref1
4xCTF-hP/DCaro4hP
















Percentage of
Mean
Percentage of
Mean
Percentage of
Mean
Percentage of
Mean


Weeks
GFP+ cells
Fluorescence
GFP+ cells
Fluorescence
GFP+ cells
Fluorescence
GFP+ cells
Fluorescence


















1
0.38%
333.41%
0.37%
280.18%
0.69%
37.40%
0.50%
24.91%


3
0.43%
1462.89%
0.30%
1556.39%
0.35%
318.98%
0.43%
164.46%


4
0.48%
1864.00%
0.41%
1072.00%
0.47%
220.11%
0.44%
112.31%


5
0.46%
1150.00%
0.40%
798.94%
0.47%
114.21%
0.44%
72.12%


6
0.43%
1095.00%
0.37%
914.00%
0.41%
117.68%
0.38%
70.89%


7
0.44%
1114.00%
0.35%
804.86%
0.44%
137.55%
0.41%
74.63%


8
0.45%
972.55%
0.42%
672.55%
0.46%
103.23%
0.44%
62.95%


10
0.44%
990.18%
0.41%
851.38%
0.49%
148.49%
0.46%
82.63%









2.8 Genotoxicity
The development of retroviral vector-induced lymphoproliferative disease in patients with X-linked severe combined immunodeficiency (SCID-X1) otherwise successfully treated by the gene therapy has highlighted the need for safer vectors with reduced regulatory cross-talk with adjacent host cell genomic DNA. Using simple human HeLa cell culture, the inventors have observed that even self-inactivating LTR based lentiviral vectors may skew clonal survival over time (unpublished data). Using a combination of genome-wide non-restrictive linear amplification-mediated PCR that circumvents the limiting use of restriction enzymes during integration site analysis, and next generation sequencing technologies (GSF1× and GSF1× Titanium; Roche Diagnostics), the inventors aimed to assess the vector biosafety level of any integrating vector type by comprehensive integration site profiling.
Comprehensive integration site analyses is performed at regular intervals after transduction of cells (e.g. 1 week, 4 weeks, 8 & 12 weeks), thereby assessing the total number of retrieved integration sites, quantitative contribution of individual clones at a given time point and over time, formation of integration site clusters (‘hotspots’ or CIS, common integration sites) and characterization of vector targeted cellular genes (overrespresented gene class categories).
Genotoxicity studies have now indicated the effectiveness of the 4×CTF insulated gammaretrovectors in reducing the probability of selecting with time dominant clones, following integration into the host target cell genome. The assessment of putative residual genotoxicity is made by comparing the evolution of integration patterns in human target cells grown and sequentially sampled over a one to three months period with both reference (non-insulated) and test insulated vectors, using high-throughput pyro-sequencing.
Data from the genotoxicity study are shown in Table 7 below and FIGS. 22A and 22B:











TABLE 7









DCaro4.23
Test1-cPPT/Lenp29
pSIN18



















IS (SC


IS (SC


IS (SC





adjusted to


adjusted to


adjusted to


Week
IS
SC
1000)
IS
SC
1000)
IS
SC
1000)



















1
544
860
633
546
2253
242
997
1592
626


2
512
955
536
626
2406
260
925
1633
566


5
320
580
552
377
2620
144
463
1251
370


6
339
853
397
427
2800
153
301
1321
228


9
 92
514
179
121
2001
60
 70
1414
50


11 
 92
1241
74
109
1457
75
 39
1701
23


12 
 74
859
86
 54
1249
43
 35
1805
19


Total unique
1736 


1927 


2624 


(ex. mappable)
(1570) 


(1734) 


(2344) 









Over the course of this study all three tested vectors showed a reduction in the total number of integration sites. When comparing the overall number of integration sites (per 1000 sequences) remaining for each vector after 12 weeks, the non-insulated constructs showed between a 2 to 4 fold reduction in insertion sites (pSIN18 reduced to 19 integration sites and Test1-cPPT/Lenp29 reduced to 43 integration), in comparison to the insulated vector DCaro4.23 which was reduced to 86 integration. Using this measurement of genotoxicity therefore the insulated vector DCaro4.23 which comprises the 4×CTF GIE showed a significantly lower level of genotoxicity than the commonly used pSIN 18 vector or the more closely related Test1-cPPT/Lenp29 vector.
With reference to FIG. 23 it can be seen that with the DCaro4.23 vector, both VCN (Vector copy number) and % of transduced cells remain stable over the twelve week period of the experiment whereas with Test1-cPPT/Lenp29 vector (Test1), VCN decreases from 2.5 to 1.5 but % of GFP+ cells fell from 70 to 40% and pSIN18, VCN decreases from 2 to 0.7 while % GFP+ fell from 60 to 20%.
This experiment establishes that with the 4×CTF insulated lentivector, starting from an identical number of integration events leads to stable transduction and expression levels over time, while no such stability is observed with control non-insulated vectors either at the genetic or phenotypic level.
With reference to FIGS. 24, 25 and 26 it can also be seen that the insulated vector Dcaro4.23 behaves in a very similar manner to pSIN18 vector (Ref1) or Test1-cPPT/Lenp29 vector (Test1) in terms of its chromosomal distribution pattern (FIG. 24) or the distribution of integration sites in or around genes (FIGS. 25 and 26). Indicating that the presence of the insulating element does not affect the inter- or intra-chromosomal integration of the vector.
Table 8 shows an analysis of the distribution of integration sites for each of the tested vectors in one or more of the common integration sites identified for each of the vectors. No obvious differences were seen between the vectors in terms of the number of integrations in the common integration sites.












TABLE 8







Vector
DCaro4.23
LenP29
pSIN18





















# IS (total)
1570
1734
2344



# CIS
82
100
183











CIS order
2nd
74
83
152



3rd
5
12
18



4th
—
3
2



5th
2
2
4



6th
1
—
2



7th
—
—
1












IS in CIS
179 (11%)
224 (13%)
405 (17%)










The data presented above indicates that although the vector comprising the insulator element shows similar patterns of integration distribution throughout the genome, whilst showing lower levels of clonal expansion associated with genotoxicity.
2.9 Transduction of Human hES Cell-Line hES Sa01
The inventors have also shown that the gene transfer vector according to the present invention can be transduced into human embryonic stem cells (hES).
The hES cell-line was transformed as follows:

    
    
        Tissue was either partially dissected into clumps or completely dissociated into a population of free cells.
        Approximately 800,000 cells were transduced using gene transfer vectors according to the present invention comprising (i) at least one insulator or (ii) no insulator, at a MOI of 50.
        Transduction occurred under orbital agitation of no more than 8 revolutions per minute at 37° C. for 6 hours, in the presence of protamine sulfate at a concentration of 5 μg/ml in a final volume of 1 mL.
        Cells are then separated into two equal groups, with the first group being plated directly and the second group being subject to trypsin digestion so as to disassociate the cells prior to plating. Both groups were then cultured using complete medium.
        Cells were then subjected to fluorescence microscopy every 3⁄4 days.
    
    


At day 6 after transduction, fluorescent cells were observed indicating effective transduction and expression of the transgene starting using the lentiviral construct DCaro4.23 and the internal promoter Fr-MuLV-U3 (FIG. 21).
The inventors have also successfully tranduced into iPS (Induced pluripotent stem) cells vectors comprising GIEs according to the present Patent Application and established effective transduction and expression of transgenes using these constructs and in particular the lentiviral vector DCaro4.23.
Example 3
Conclusions
SIN-insulated retrovectors have been constructed with two candidate GIEs and compared to native and SIN-LTRs. With each constructs two internal promoters have been tested: either the strong Fr-MuLV-U3 or the housekeeping hPGK. The inventors have identified a specific combination of insulator 2 repeats which shows functional activity, high titres and boundary effect in both gammaretrovectors (p20) and lentivectors (DCaro4).
In target cells a dramatic shift of expression is observed with an homogenous expression profile, the level of which is linked to the promoter strength. These data remain stable in both HeLa cells and cord blood HSCs for over three months, irrespective of the multiplicity of infection (MOI). In comparison, control vectors show heterogeneous expression profiles with levels which depend on the MOI and prove unstable over time. The inventors have undertaken genotoxicity assessments in comparing integration patterns in human target cells sampled over three months using high-throughput pyro-sequencing. Preliminary data indicate a flat pattern without CIS meaning the vectors according to the present invention have another advantage with respect to prior art vectors pertaining to enhancer-blocking activity harbouring potential for reduced genotoxicity.
The inventors sought to design synthetic GIEs consisting of multimers of critical core sequences. The first insulator they designed consists of a new combination of CTCF binding sites: 42 bp FII sequence (CTCF1) derived from HS4 and 39 bp BEAD-A sequence (CTCF2) derived from the human T cell receptor alpha/delta locus and is known as Insulator 1. Insulator 1 consists of three repeats of the CTCF1-CTCF2 sequence.
The second insulator consists of multimerised CTF sites with various numbers of repeats. CTF otherwise known as a transcription factor, has recently been shown to act as a chromatin domain boundary that shields human telomeric genes from silencing.
When either of the two insulators were cloned into the LTR of simple gamma retroviral SIN constructs, virus production dramatically dropped, as compared to reference vectors with either full or SIN-LTRs. The inventors were able to identify key determinants which allow the production of insulated SIN gamma retroviral vectors with good titers.
Among these, a key sequence is the 3′ untranslated region (3′UTR) derived from the Fr-MuLV FB29 strain which features have already been reported in full-LTRs recombinant retroviral vectors (Cohen-Haguenauer et al, 1998). The addition of this sequence caused a dramatic increase in the virus titers obtained with the insulated vectors; those containing Insulator 1 had titers increased by one order of magnitude while with Insulator 2, titers ranged across several log.
With the other element, a human-beta globin derived, synthetic polyA sequence was placed downstream of the vector 3′LTR, in order to facilitate the synthesis of full-length RNA transcripts to be packaged into virions, a paradoxical effect was observed, titers of Insulator1 containing constructs dramatically dropped while for constructs containing Insulator 2, titers increased by 3 to 5 fold.
These first observations showed significant differences between Insulators 1 and 2 and were verified by further experiments which were independently reproduced several times and led to identical data.
With insulator 1: (i) recombinant viruses are produced at much lower titers than controls, although rescued by the addition of Fr-MuLV 3′UTR and (ii) overall expression levels are also much lower as compared to full LTR vectors. The opposite is observed with Insulator 2, since both titers and expression levels are higher than with control vectors. It is noteworthy that with lentivectors, these same observations hold true, except that in primary human haematopoietic stem cells in which Lenp29 vector has a unique potential in terms of infectivity, with a higher percentage of transduced cells as compared to pSIN-18 and DCaro4.23, confirming prior observations.
In addition, an important observation has been made following long term monitoring of transduced and sorted eGFP positive HeLa cells which has been sustained over a 120 days period, alsoaiming at genotoxicity studies. Although the level of eGFP expression remained stable over time in each sample studied, a drop in the percentage of eGFP positive cells was gradually observed in p11 transduced cells, starting from day 30 and progressively dropping over time, from 90 to 30%.
Based on these observations, Insulator 2 has characteristics which appear to make it the most useful insulator element identified and because of this the inventors performed further experiments to determine the optimum number of repeats. Gammaretroviral vectors were constructed where either 2 copies, 4 copies or 7 copies of Insulator 2 in the SIN-virus LTR. Comparative experiments with these vectors indicate that the best levels of eGFP fluorescence are observed with 4×CTF, in comparison to either 2×CTF or 7×CTF vectors. In addition, both virus titres and expression levels of the reporter gene in target cells tend to be lower with 7×CTF vectors in comparison to 2×CTF vectors.
Similar data were obtained with a set of lentivectors with either 4× Ins2 (DCaro4.23) or 8× Ins2 (DCaro8.22), wherein titers differ by at least one log range in favour of DCaro4.23. In addition, when the multiplicity of infection (MOI), that is the ratio of infectious virus to cells is normalised, the infectious potential capacity to express the transgene following transduction and integration of insulated vectors, is much higher with DCaro4.23.
Finally, in primary haematopoietic CD34+ cells from cord blood, DCaro4.23 performed with characteristics fulfilling our initial requirements. The inventors search for a putative gene therapy vector with decreased genotoxicity levels but no drop in integration efficacy has therefore resulted in the lentivirus construct DCaro4.23. Further aspects of the behaviour of Dcaro4.23 in particular when the FR-MuLV 3′UTR is included remain to be investigated and could potentially increase the utility of this vector further, such further constructs would also require validation particularly in terms of its genotoxicity. In addition the gammaretrovirus vectors p19 and p20 also represent putative gene therapy constructs.
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## Claims

1. An integrating gene transfer viral vector (IGTV) which comprises at least one genetic insulator element (GIE), wherein said at least one GIE comprises at least two copies of an element selected from the group consisting of (A) a CTF binding site; and (B) a sequence consisting of a first CTCF binding site and a second CTCF binding site: wherein the CTF binding site is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; and wherein said first and said second CTCF binding sites are from regulatory sequences of different genes and wherein said first and second CTCF binding sites comprise a different sequence selected from the group consisting of SEQ ID:7, SEQ ID NO:8, SEQ ID NO:27, and SEQ ID NO:31,
wherein the at least one GIE is positioned in the vector such that following genomic insertion of the IGTV, the at least one GIE is positioned between a genomic sequence and the coding sequence of the IGTV.

2. The IGTV according to claim 1, wherein the at least one GIE comprises at least two copies of said sequence consisting of said first and second CTCF binding site and comprises a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 27 and SEQ ID NO: 31.

3. The IGTV according to claim 1, wherein said at least one GIE comprises the sequence SEQ ID NO: 6.

4. The IGTV according to claim 1, wherein the at least one GIE comprises said CTF binding site selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30.

5. The IGTV according to claim 4, wherein said GIE comprises between two and eight copies of said CTF binding site.

6. The IGTV according to claim 1, wherein said IGTV is a retrovirus vector.

7. The IGTV vector according to claim 6, wherein IGTV is self-inactivating following insertion into a genome.

8. The IGTV according to claim 1, which further comprises an exogenous enhancer.

9. The IGTV according to claim 8, wherein said exogenous enhancer is positioned between said GIE and an exogenous transgene encoded by said IGTV.

10. The IGTV according to claim 9, wherein said enhancer is selected from the group consisting of viral enhancers, eukaryotic enhancers, animal enhancers, mammalian enhancers.

11. The IGTV according to claim 8, wherein said enhancer comprises the sequence SEQ ID NO: 22.

12. The IGTV according to claim 1, which further comprises a polyA tail.

13. A cell, comprising the IGTV according to claim 1.