Patent Application
20200199619
The present invention refers to improved gene transfer vectors for gene therapy and basic research. Particularly, the present invention relates to new DNA sequences capable of improve expression regulation and avoid silencing of gene transfer vectors (viral or non-viral). More particularly, the present invention refers to new combinations of DNA sequences capable of improve expression regulation and avoid silencing of gene transfer vectors (viral or non-viral) in mammalian stem cells. More particularly, the present invention refers to new combinations of DNA sequences capable of improve expression regulation and avoid silencing of gene transfer vectors (viral or non-viral) for gene therapy applications.
Technologies allowing conditional transgene expression in human stem cells are fundamental not only to study gene function but also as potential tools for gene therapy. Gene therapy has been proposed to be a promising tool to treat diseases. The aim of gene therapy is to introduce therapeutic genes into target cells, leading to efficient, stable and/or regulated expression of the therapeutic molecules and without affecting the normal physiology of the target cell.
However, gene transfer vectors (the tools used to transfer the gene into the cells) are confronted with several obstacles such as:
Several approaches have been proposed to try to solve these obstacles, such as the use of human promoters (Hong, S. et al. Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells. Mol Ther (2007); Kita-Matsuo, H. et al. Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. PLoS ONE (2009)), or of promoters with low methylation levels, or the incorporation of insulators (Benabdellah, K. et al., A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells. PLoS ONE (2014); Aker, M. et al. Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum Gene Ther (2007); Pfaff, N. et al. A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny. Stem Cells (2013)) in order to avoid the deleterious effects of the transgene over the host chromatin and viceversa. The insulators are based on naturally occurring DNA elements that form functional boundaries between adjacent chromatin domains and play a role in shielding certain genes from other regulatory domains present on its proximity. However, each of these strategies alone is not sufficient to completely solve the obstacles.
In addition, the target cell also dictates the stability of transgene expression and the potential genotoxicity of the integrative vector. In this sense, transgene expression on stem and primary cells are generally more prompt to gene silencing than on differentiated and/or immortalized cell lines.
Tetracycline-regulated gene expression systems (Tet-On or Tet-Off) have been used for conditional gene expression in most stem cells types including human embryonic stem cells (hESCs) (Ormsbee Golden, B. D. et al., Sox2 expression is regulated by a negative feedback loop in embryonic stem cells that involves AKT signaling and FoxO1. PLoS ONE (2013); Vieyra, D. S. & Goodell, M. A. Pluripotentiality and conditional transgene regulation in human embryonic stem cells expressing insulated tetracycline-ON transactivator. Stem Cells (2007); Zhou, B. Y. et al. Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer. Stem Cells (2007); Xia, X. et al., In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives. Stem Cells (2008)), induced pluripotent stem cells (iPSCs) (Qian, K. et al. A simple and efficient system for regulating gene expression in human pluripotent stem cells and derivatives. Stem Cells (2014)) and mesenchymal stromal cells (hMSCs) (Pan, Z. M. et al. Treatment of Femoral Head Necrosis With Bone Marrow Mesenchymal Stem Cells Expressing Inducible Hepatocyte Growth Factor. Am J Ther (2015); Moriyama, H. et al. Tightly regulated and homogeneous transgene expression in human adipose-derived mesenchymal stem cells by lentivirus with tet-off system. PLoS ONE (2013); Yang, W. H. et al. Regulated expression of lentivirus-mediated GDNF in human bone marrow-derived mesenchymal stem cells and its neuroprotection on dopaminergic cells in vitro. PLoS ONE (2013)).
However, most tetracycline-regulated systems require a tetracycline-dependent-transactivator containing the activating domain of the herpes virus simplex viral protein 16 (VP-16) linked to the TetR repressor (tTA for Tet-Off or rtTA for Tet-On systems). The presence of this transactivating domain can make these proteins toxic due to the sequestration of transcription factors required for cell growth (squelching) (Sisson, T. H. et al. Expression of the reverse tetracycline-transactivator gene causes emphysema-like changes in mice. Am J Respir Cell Mol Biol (2006); Pen, A. K. et al., Conditional expression of genes in the respiratory epithelium in transgenic mice: cautionary notes and toward building a better mouse trap. Am J Respir Cell Mol Biol (2009)). In addition, binding of the chimeric TetR-VP16 protein to pseudo-TetO sites can trans-activate non-target cellular genes (Hackl, H. et al., Tetracycline regulator expression alters the transcriptional program of mammalian cells. PLoS ONE (2010); Han, H. J. et al. Strain background influences neurotoxicity and behavioral abnormalities in mice expressing the tetracycline transactivator. J Neurosci (2012)) causing unpredicted side effects. Similar consequences have also been reported with other transcriptional activators such as the Cre-recombinase and its variant CreER (Forni, P. E. et al. High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly. J Neurosci (2006); Higashi, A. Y. et al. Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2. J Immunol (2009); Naiche, L. A. & Papaioannou, V. E. Cre activity causes widespread apoptosis and lethal anemia during embryonic development. Genesis (2007)). Therefore, even though the tTA (rtTA)/tetO and Cre/loxP systems are useful tools for conditional transgene expression in stem cells, they have the potential to influence cellular physiology. Another major obstacle for the wider application of most conditional systems is the general requirement of a selection of the cells that have integrated the transgenes, mainly due to the low efficiency of gene delivery methods and the strong silencing of the transgenes. Cells then have to be exposed to additional steps that add potential side effects to the method and clones might be drug-responsive while being able to regulate transgene expression.
The inventors has described an all-in-one regulated lentiviral vector (CEST) based on the original TetR repressor, that does not need the tetracycling-transactivating domain and that allowed the generation of Dox-regulated cell lines, including primary human fibroblasts (HFF) and human MSCs (hMSCs). However, the CEST LVs were not able to efficiently regulate transgene expression in pluripotent stem cells due to silencing of the TetR expression. In addition this system required multiple integrations per cell in order to achieve regulation in HFF and hMSCs, hindering its application for gene therapy applications.
The inventors have also recently developed a chimeric insulator (Is2) that avoid silencing of LVs in pluripotent hESCs during expansion and upon differentiation (Benabdellah, K. et al., A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells. PLoS ONE (2014 and patent EP13382080.3).
Overall, although significant improvements have been done in the past years to develop efficient transgene systems, additional efforts are needed to improve the existing techniques and to reach higher efficiency of transgene insertion, transgene expression and expression regulation in stem cells.
In the present invention, the inventors have generated a Tet-On all-in-one construct that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the integrative construct with the Is2 insulator, they have constructed the Lent-On-Plus Tet-On system that achieves efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus system did not require selection or cloning, and transgene regulation is maintained after long-term cultured and upon differentiation toward different lineages.
The present invention offers an alternative solution for the problem cited above, of a need to improve transgene expression systems in stem cells. As shown herein, several genetic constructs have been tested on their capacity to induce the expression of reporter gene (eGFP) upon doxycycline stimulation on the context of a CEST Lentiviral Vector (LV) or of a CEET LV. Both vectors are based on the Tet-On system.
The CEST LV contains a CMV-TetO inducible promoter that controls the expression of the reporter gene eGFP. A spleen focus forming virus (SFFV) promoter controls the expression of a TetR gene that encodes a protein based on the TetR repressor. Upon binding to the CMV-TEtO promoter, the TetR protein inhibits the expression of the eGFP reporter gene. In the presence of doxycycline, the TetR protein is no longer able to repress the CMV-TetO promoter.
The CEET vector is similar to the CEST LV except that it contains a human elongation factor-1 alpha (hEF1α) promoter instead of the SFFV to control the expression of the TetR protein. The efficiency of eGFP expression in the presence and absence of doxycycline is tested for both vectors, in different mammalian host cells, when containing additional sequences: insulator Is2 with or without the nuclear localization signal nl2 associated to the TetR protein.
In 293T cells, the addition of nuclear localization signal (nl2) to the TetR protein in the CEST vector increased the expression of eGFP. Both, the addition of the nl2 and IS2 to the CEET vector improved the fold induction of eGFP upon doxycycline stimuli. This combination of elements (nl2, hEF1α and Is2) also reduced the unspecific expression of eGFP in the absence of doxycycline resulting from leaking. These improvements were obtained in conditions of low multiplicity of infection (MOI) that mainly allow to transducing only one vector copy per cell.
Similarly, in human mesenchymal stem cells (hMSCs), the use of the CEET vector instead of the CEST vector reduced the leaking in a construct already containing the nl2 signal. The addition of the Is2 to this construct also improved the induced expression of the transgene. This tendency was maintained upon long term culture and differentiation. Again, these results were also observed with cells harboring only one vector copy per cell.
In human embryonic stem cells (hESCs), the use of the CEET vector in combination with the nl2 with or without the Is2 allowed to reach low leaking and good transgene expression in the presence of doxycycline. Upon differentiation towards hMSCs and towards hematopoietic stem cells, the low leaking and good expression levels of the previous constructs was maintained. However, the beneficial effect of the Is2 was mainly observed in the differentiated hMSCs.
Taken together, results show that the use of the hEF1 α promoter, instead of by the SFFV promoter, the addition of the nuclear localization signal nl2 to the TetR protein and the addition of the insulator element IS2 allow to reduce the unspecific expression of the transgene due to leaking and to improve its regulation upon doxycycline stimulation in different human stem cell from different origins (including pluripotent hESCS) even with low multiplicity of infection (MOI). Overall, the present invention offers an improved method to express and regulate the expression of a transgene in different types of human stem cells including pluripotent stem cells.
(a) Representative plots showing eGFP expression profiles of untransduced 293T (Mock) and 293T cells transduced with the different LVs (as indicated at the top of each plot) in the absence (top) or presence (bottom) of Dox (0.1 μg/ml). A MOI=0.3 was used to keep the percentage of eGFP+ cells below 30% (in order to keep transduced cells with only one LV integration). The gates of the eGFP+ populations were set to 0.2-0.4% of eGFP+cells in the untransduced population (Mock; left plots) and subtracted to the % obtained under the different vectors and conditions for the analysis. The percentage (%) of the eGFP+ population (used to measure leaking) and the Mean Fluorescence Intensity (MFI) of the eGFP+ population are shown in each plot (b). Graph showing leaking of the different LVs in 293T taking into account the MFI of the eGFP+ population in the absence of Dox: (Leaking=[[% eGFP+(−Dox)*100/% eGFP+(+Dox)]*MFI eGFP+(−Dox)]. To measure leaking, the background (% of eGFP+ of Mock cells) were subtracted to the % of the eGFP+ under the different conditions. Values represent mean +/−standard error of the mean of at least four separate experiments Asterisks indicate significance related to CEST (on top of the bars) or significance between the CEETnl2 and CEETnl2Is2 (as indicated in the Figure) (*p<0.05; **p<0.01).
Additionally, in some cases gene expression leads to the production of other forms of RNA that are not translated into proteins, such as for example transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), long non-coding (IncRNAs), or RNAs that will derive into micro-RNAs (miRNAs) or small interfering RNAs (siRNAs).
The present invention offers a genetic construct capable to introduce a specific gene cassette into the genome of a target cell, that promotes a better regulation of a transgene and that requires a lower MOI, compared to previous constructs of the same type. Additionally, this construct allows for significant expression of the transgene in different human stem cells where previous constructs show no regulation of expression. Therefore, the present invention offers a solution to the need of technical improvements to reach tightly regulated transgene expression in bulk populations after one single round of transduction/transfection, especially for transgenes to be introduced in human stem cells.
The first aspect of the present invention refers to a genetic construct having nucleic acid sequences capable of integrating into the genome of a mammalian cell comprising:
a) two regulatory control elements, b) at least two coding nucleic acid molecules referred to as the first coding nucleic acid molecule and the second nucleic acid molecule, or as NAm-A and NAm-B respectively, operatively associated with the regulatory control elements and capable of expression in the cell, c) a nucleic acid molecule encoding a nuclear localization element, and d) an insulator element,
wherein NAm-B encodes a protein capable of repressing the regulatory control element, from now on referred as RCE-A, that controls the expression of NAm-A, wherein RCE-A is inducible and can be regulated by the protein encoded by NAm-B, wherein the nucleic acid molecule encoding the nuclear localization signal is associated to NAm-B so that both nucleic acid molecules encode a single polypeptide.
The nucleic acid molecule encoding the nuclear localization signal preferably encodes the nuclear localization signal from the glucocorticoid receptor, referred to as nuclear localization signal 2 (nl2), preferably with nucleic acid sequence SEQ ID n° 4 (gcccgaaaatgtcttcaggctggaatgaacctggaagctcgaaaaacaaagaag). The insulator preferably consists on the SEQ ID n° 1, even more preferably consists on the SEQ ID n° 1 associated with SEQ ID n° 2 so that they can form a single nucleic acid sequence, yet more preferably consists on the SEQ ID n° 3. In a preferred embodiment, the nucleic acid molecule encoding the nuclear localization signal is SEQ ID n° 4 and the insulator the one with SEQ ID n° 3.
In previous embodiments of the invention, suitable sequences preferably have at least about: 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, over the total length of the sequence, to the sequence used to define them. Identity refers to the similarity of two nucleotide sequences that are aligned so that the highest order match is obtained. Identity is calculated according to methods known in the art. For example, if a nucleotide sequence (called “Sequence A”) has 90% identity to a portion of SEQ ID NO 1, then Sequence A will be identical to the referenced portion of SEQ ID NO 1 except that Sequence A may include up to 10 point mutations (such as substitutions with other nucleotides) per each 100 nucleotides of the referenced portion of SEQ ID NO 1.
The invention also includes an insulator that has a nucleic acid sequence with sufficient identity to the insulator elements described previously in this section of the invention to hybridize with them under stringent hybridization conditions. In this sense, the present invention also includes insulator elements having nucleic acid molecules that hybridize to one or more of the sequences in SEQ ID n° I-SEQ ID n° 3 or its complementary sequence. Similarly, the invention also includes a nucleic acid sequence with sufficient identity to the nuclear localization signals described previously in this section to hybridize with them under stringent hybridization conditions. In this sense, the present invention also includes insulator elements having nucleic acid molecules that hybridize to SEQ ID n° 4 its complementary sequence. Such nucleic acid molecules preferably hybridize under high stringency conditions (see Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). High stringency washes preferably have low salt contents (preferably about 0.2% SSC) and a temperature of about 50-65° C. Thus, in another embodiment, the insulator element and/or the nucleic acid molecule encoding the nuclear localization signal are introduced in the anti-sense orientation. Additionally, other insulator elements and/or nucleic acid molecules encoding other nuclear localization signals can be readily inserted in the genetic construct provided that expression of NAm-A and NAm-B still occurs and that the encoded nuclear localization signal promotes the import of NAm-B encoded protein into the nucleus of the host cell.
Commonly, the purpose of introducing a transgene in a host cell is to promote its expression in this cell, and ideally it should be possible to turn on and off its expression according to the needs. Since the protein encoded by NAm-B can activate or repress the expression of NAm-A, ideally this activator or repressor function on NAm-A expression might be regulatable according to the needs. A common mechanism of regulation is that NAm-B protein can be targeted by a molecule in the cell, that is itself present in the cell according to the needs. Therefore, in a preferred embodiment, NAm-B encodes a protein whose regulatory function is drug-responsive, preferably tetracycline—or a variant thereof—responsive. In a more preferred embodiment, the regulatory element is based on the original TetR repressor element. In the context of the present invention, it is understood that the original TetR repressor is the product of the TetR gene (the TetR protein) from Escherichia coli.
In order to regulate the expression of NAm-A, NAm-B encoded protein has to be able to bind to RCE-A that controls the expression of NAm-A. Thus, in another embodiment, the RCE-A comprises a binding site for the protein encoded by NAm-B. In addition, preferably the regulation of NAm-A expression by NAm-B encoded protein is as follows: in the absence of the drug described in the previous paragraph, binding of NAm-B encoded protein to RCE-A leads to the repression of RCE-A and to the inhibition of NAm-A expression. Thus in another preferred embodiment, NAm-B encodes a protein that binds to REC-A to inhibit the expression of NAm-A. Preferably, in the presence of the drug, Nam-B encoded protein cannot further repress the expression of NAm-A. Then, in a yet more preferred embodiment, in the presence of the drug described previously in this section, NAm-B cannot efficiently repress the expression of NAm-A. Yet, in another more preferred embodiment, RCE-A comprises the Cytomegalovirus (CMV) promoter.
To allow that the molecule that regulates the function of the NAm-B encoded protein (such as a tetracycline-derived drug) has an effect on NAm-A expression at any time, the expression of NAm-B might be constitutive. Thus, in another preferred embodiment, the regulatory element that controls the expression of NAm-B (from now on referred as RCE-B) is a constitutive promoter. Preferably, RCE-B is the spleen focus forming virus (SFFV) promoter. According to
Overall, in a more preferred embodiment, the genetic construct of the first aspect of the invention comprises: an RCE-A that contains a binding site for the regulatory protein encoded by NAm-B and preferably further comprises the CMV promoter; an RCE-B that is constitutively active, preferably that is the SFFV promoter, more preferably that is the hEF1α promoter; a NAm-B that encodes a protein whose function is drug-responsive, preferably tetracycline—or a variant thereof—responsive, more preferably is based on the TetR repressor element, yet more preferably it represses the expression of NAm-A in the absence of the drug; a nucleic acid molecule (associated with the NAm-B so that both nucleic acid molecules encode a single polypeptide) that encodes a nuclear localization signal, preferably encoding that of the glucocorticoid receptor referred as nl2, more preferably sharing at least about a 70% of identity over the total length of the sequence with the sequence ID SEQ n° 4, yet more preferably it is the sequence ID SEQ n° 4; wherein the insulator sequence is the one sharing at least about a 70% of identity over the total length of the sequence preferably with the one consisting on the SEQ ID n° 1, even more preferably with the one consisting on the SEQ ID n° 1 associated with SEQ n° 2 so that they form a single nucleic acid molecule, yet more preferably with the one consisting on the SEQ ID n° 3; more preferably, the insulator sequence is any of the ones just mentioned, more preferably it is SEQ ID n° 3.
The final goal of gene transfer is to insert a gene into a cell (and ultimately into an organism) using a vector that will transfer only the required nucleic acid sequences into the desired target cells. Viruses are naturally very efficient at transducing their own genetic information into host cells for their own replication. By replacing non-essential viral genes with foreign genes of therapeutic interest, recombinant viral vectors can be used to transduce the cell type that they would normally infect. For biosafety and viral safety reasons, in addition to the transgene(s) of interest, these modified vectors in many cases contain/express only the viral sequences required for the initiation of viral DNA replication and packaging (Bouard D. et al., Viral vectors: from virology to transgene expression, British Journal of Pharmacology (2009)). Therefore, in a particular embodiment, the genetic construct according to any of the previous embodiments is a viral vector. Examples of viral vectors that are commonly used for this purpose are retrovirus vectors, lentivirus vectors, Adenovirus Vectors, Adeno-associated virus (AAV) vectors and Semliki Forest Virus.
Retroviruses are viruses that carry RNA as genetic material instead of DNA and usually also carry reverse transcriptase enzymes that convert RNA into its DNA copy. Then, this DNA is integrated into the host genome using integrase enzyme. Thus, retroviruses are a subtype of virus that integrate their own genetic information into the host cell genome. They are broadly used in different gene transfer techniques. Therefore, in a preferred embodiment the genetic construct of the present invention is a retroviral vector.
Retrovirus vectors contain 5′ and 3′ Long Terminal Repeats (LTRs) that are found at either end of proviral DNA formed by reverse transcription of retroviral RNA. They are used by retroviruses to insert their genetic material into the host genomes. Each LTR consists of a U3, R, and U5 region. The ends of the LTRs subsequently participate in integration of the provirus DNA into the host genome. Once the provirus has been integrated, the LTR on the 5′ end serves as the promoter for the entire retroviral genome, while the LTR at the 3′ end provides for nascent viral RNA polyadenylation. The 3′ LTR usually acts in transcription termination and polyadenylation. For safety reasons, retroviral vectors have the LTR promoter (U3) deleted and use an internal promoter to express transgenes in transduced cells.
Lentiviruses are a subtype of retroviruses. They present several advantages compared to other retroviruses for gene transfer purposes: (i) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (ii) contrary to most retroviruses, the capability of infecting both dividing and non-dividing cells; (iii) no expression of viral proteins after vector transduction; (iv) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (v) potentially safer integration site profile; and (vi) a relatively easy system for vector manipulation and production (Sakuma T. et al., Lentiviral vectors: basic to translational, Biochemical Journal (2012)). Therefore, they have been used to transfer genes into delicate cells such as stem cells or neurons. (Miyoshi H et al., Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science (1999); Lois C et al., Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science (2002)). Thus, in a more preferred embodiment, the genetic construct of the present invention is a lentivirus vector (LV).
A second aspect of the invention refers to a composition comprising the genetic construct according to any of the previous embodiments, wherein this composition can be a pharmaceutical composition (from here in after pharmaceutical composition of the invention). The pharmaceutical composition of this invention can be used to treat patients having diseases, disorders or abnormal physical states such as blood diseases or neural diseases (neurodegenerative). Blood diseases treatable by stem cell transplant include leukemias, myelodysplastic syndromes, stem cell disorders, myeloproliferative disorders, lymphoproliferative disorders phagocyte disorders, inherited metabolic disorders, histiocytic disorders, inherited erythrocyte abnormalities, inherited immune system disorders, inherited platelet abnormalities, plasma cell disorders, and malignancies. Stem cell nerve diseases to be treated by neural stem cell transplantation include diseases resulting in neural cell damage or loss, eg. paralysis, Parkinson's disease, Alzheimer's disease, ALS, multiple sclerosis. Thus, in a particular embodiment of the second aspect, the invention is for use in therapy. It also relates to a method of medical treatment of a mammal, preferably a human, by administering to the mammal a genetic construct according to any of the previous embodiments of the invention or a cell containing this construct. The pharmaceutical composition of the invention can be administered by ex vivo and in vivo methods such as electroporation, DNA microinjection, liposome DNA delivery. Derivatives or hybrids of these vectors may also be used. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration.
The pharmaceutical composition of the invention could include acceptable carriers or excipients. It can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the genetic construct is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA). On this basis, the pharmaceutical composition could include an active compound or substance, such as the genetic construct, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isosmotic with the physiological fluids. The methods of combining the genetic construct with the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the transport of the active compound to specified sites within the target cells. Thus, in a particular embodiment of the invention, the pharmaceutical composition optionally comprises a pharmaceutically acceptable vehicle.
In addition, the present invention also includes compositions and methods (from herein referred as method of the invention) for providing a genetic construct as defined in any of the embodiments of the first aspect of the invention, to a subject such that expression of the nucleic acid molecules of the genetic construct (NAm-A and NAm-B) in the cells provides the biological activity of the polypeptide encoded by the coding nucleic acid molecules in those cells. The invention includes methods and compositions for providing a coding nucleic acid molecule NAm-A to the cells of an individual such that the expression regulation of NAm-A in the cells provides the biological activity or phenotype of the polypeptide encoded by NAm-A to the cell. The method also relates to a method for providing an individual having a disease, disorder or abnormal physical state with a biologically active polypeptide by administering a genetic construct of the present invention. The amount of polypeptide will vary with the subject's needs. The optimal dosage of genetic construct may be readily determined using empirical techniques, for example by escalating doses. Various approaches to gene therapy may be used. The invention includes a process for providing a human with a therapeutic polypeptide including: introducing human cells into a human, said human cells having been treated in vitro or ex vivo to insert therein a pharmaceutical composition of the invention or a genetic construct of the invention, the human cells expressing in said human a therapeutically effective amount of said therapeutic polypeptide.
The method also relates to a method for producing a stock of recombinant virus by producing virus suitable for gene therapy. This method preferably involves transfecting cells permissive for virus replication and collecting the virus produced.
Furthermore, the invention also relates to a mammalian host cell (isolated cell in vitro, a cell in vivo, or a cell treated ex vivo and returned to an in vivo site) comprising the genetic construct of any of the embodiments of the first aspect of the invention. In this sense, cells transfected with a nucleic acid molecule as a DNA molecule, or transduced with the nucleic acid molecule as a DNA virus vector, may be used, for example, in bone marrow or cord blood cell transplants according to techniques known in the art. Examples of the use of transduced bone marrow or cord blood cells in transplants are for ex vivo gene therapy of Adenosine deaminase (ADA) deficiency. Other cells which may be transfected or transduced either ex vivo or in vivo include purified stem cells. Thus, a third aspect of the invention refers to a mammalian host stem cell comprising the genetic construct according to any of the embodiments of the first aspect of the invention. In a particular embodiment, the host stem cell is a cell of embryonic or adult tissue origin.
In any case such a mammalian cell or mammalian host cell transfected or transduced with the genetic constructs can be useful as research tools to measure levels of expression of the coding nucleic acid molecule NAm-A and the activity of the polypeptide encoded by NAm-A. In this sense the genetic constructs of the invention are useful in research to deliver marker genes or antisense RNA to cells. In a particular embodiment, the invention refers to the use of the genetic construct according to any of the embodiments of the first aspect of the invention, for cell marking. In another particular embodiment, the invention refers to the use of the genetic construct according to any of the previous embodiments, for cell genetic manipulation studies.
A fourth aspect of the invention refers to a method for expressing a nucleic acid molecule in a mammalian host cell, comprising a) administering to the cell an effective amount of the genetic construct according to any of the previous embodiments, and b) expressing the nucleic acid molecules of the genetic construct to produce the coding nucleic acid molecule RNA and its encoding polypeptide. Preferably, the host cell is a stem cell.
A fifth aspect of the invention refers to a method for producing a polypeptide in a mammalian host cell, comprising a) administering to the cell an effective amount of the genetic construct according to any of the previous embodiments, and b) expressing the nucleic acid molecule of the genetic construct to produce the coding nucleic acid molecule RNA and its encoding polypeptide. Preferably, the host cell is a stem cell.
In a preferred embodiment of the fourth and fifth aspects of the invention, the host cell is a pluripotent stem cell of embryonic origen (ESCs) or adult tissue origin (iPS). In a particular embodiment, the host cell is a cell factory for protein production (ie CHOs, HEK free style).
Finally a further aspect of the invention is an isolated polypeptide produced from a genetic construct or vector of the invention according to a method of the invention.
The following examples are only meant for illustrative purposes.
Plasmids Construction
Two different nuclear sequences named nl1 and nl2 were incorporated at the 3″end of TetR preceding the stop codon in the StetR LV (Benabdellah, K. et al., Development of an all-in one lentiviral vector system based on the original TetR for the easy generation of Tet-ON cell lines. PLoS ONE (2011)) to generate STetRnl1 and STetRnl2. nl1 consist in a three tandem repeat sequence corresponding to the nuclear localization signal (DPKKKRKV) from SV40, whereas nl2 (RKCLQAGMNLEARKTKK) is the nuclear localization signal from the glucocorticoid receptor. A Pstl-Pstl 1231 bp fragment from the STetRnl1 and STetRnl2 LVs was inserted within the unique Pstl site of the CEWP vector (Demaison, C. et al. High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther (2002)) to get the CESTnl1 and CESTnl2 respectively. CESTnl2Is2 were constructed by inserting a Kpnl-Nhel 1509 bp fragment from the SE-Is2Rev (Benabdellah, K. et al., A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells. PLoS ONE (2014)) into the Kpnl-Nhel site of CESTnl2. CEETnl2 and CEETnl2Is2 were constructed in three steps: 1—a DNA fragment encompassing the TetRnl2 sequence was amplified from the StetRnl2 and inserted into the pLVTHM LV (Addgene plasmid 12247) using standard molecular techniques to obtain the pLVTHM-TetRnl2. 2—The CEETnl2 vector was generated by insertion of a Sall-Kpnl fragment (encompassing the hEF1α-TetRnl2) into the Pstl site of CEWP. 3—Finally, the CEETnl2Is2 was constructed by inserting the Kpnl-Nhel 1509bp fragment from the SE-Is2Rev Benabdellah, K. et al., A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells. PLoS ONE (2014) into the Kpnl-Nhel site of CEETnl2.
Lentiviral vector production, cells transduction and calculation of vector copy number per cell. The human immunodeficiency virus (HIV) packaging (pCMV_R8.91) and VSV-G (pMD2.G) plasmids (http://www.addgene.org/Didier_Trono) are described elsewhere (Zufferey, R. et al., Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. (1997); Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science (1996)). Vector production was performed as previously described (Benabdellah et al., 2011). Briefly, 293T cells were plated on amine-coated petri-dishes (Sarsted, Newton, N.C.). The vector (CEST, CESTnl1, CESTnl2, CESTnl2Is2, CEETnl2 or CEETnl2Is2), the packaging (pCMVΔR8.9) and the envelope (pMD2.G) plasmids were transfected into the 293T cells using LipoD293 (SignaGen, Gainthersburg, Md., USA). Viral supernantants were collected and frozen or concentrated by ultrafiltration at 2000 g and 4° C., using 100 Kd centrifugal filter devices (Amicon Ultra-15, Millipore, Billerica, Mass.). Freshly collected virus particles, were used to transduce 293T, hMSCs and hESC as previously described (Benabdellah, K. et al., Development of an all-in-one lentiviral vector system based on the original TetR for the easy generation of Tet-ON cell lines. PLoS ONE (2011); Munoz, P. et al. Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors. PLoS ONE (2012)) Briefly, hMSCs were dissociated and mixed with the viral particles at the desired MOI, left at room temperature for 10 minutes, seeded and maintained at the incubator (5% O2; 5% CO2; 37° C. and humidity) for 5 hours. hMSCs were then washed and seeded in a bigger flask for expansion. hESCs were dissociated with collagenase type IV and plated on fresh matrigel in the presence of the fresh viral particles. The media was changed after 5 hours. When the colonies reached confluence they were split and expanded in fresh matrigel tissue flasks.
The vcn/c of transduced cells was calculated using 0.6 μg of genomic DNA (=100,000 hESCs) and plasmid DNA (from 102 to 107 copies) for the standard curve. The Q-PCR was performed as previously described (Aker, M. et al. Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum Gene Ther (2007)) using the eGFP primers shown in Supplementary Table 1.
Cell Lines and Culture Media
293T cells (CRL11268; American Type Culture Collection; Rockville, Md.) were grown in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Edinburgh, Scotland) with GlutaMAX™ and supplemented with 10% Fetal Bovine Serum (FBS) (PAA Laboratories GmbH, Austria). hMSCs were obtained from Biobanco del Sistema Sanitario Péblico de Andalucia (Granada, Spain) and cultured as previously described (Carrillo-Galvez, A. B. et al. Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity. Stem Cells (2015)). Briefly hMSCs were cultured in advanced Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, Calif.), Glutamax (GIBCO, Grand Island, N.Y., www.lifetechnologies.com), and 100 μU/ml penicillin/streptomycin (GIBCO). All experiments using human samples were performed according to the Institutional Guidelines and approved by the ethics committee of the H.U. Virgen de Macarena.AND-1 (Spanish Stem Cell Bank.)75 and H9 (Wicell Research Institute Inc. Madison, Wis.) hESC lines were maintained undifferentiated in a feeder-free culture as previously described (Munoz, P. et al. Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors. PLoS ONE (2012)). Briefly hESCs were culture in matrigel-coated T25 flasks and fed daily with hMSC-conditioned medium (Ramos-Mejia, V. et al. Maintenance of Human Embryonic Stem Cells in Mesenchymal Stem Cell-Conditioned Media Augments Hematopoietic Specification. Stem Cells Dev (2012)) supplemented with 8 ng/ml bFGF (Miltenyi Biotech, Bergish Gladbach, Germany). Approval from the Spanish National Embryo Ethical Committee was obtained to work with hESCs.
Adipocyte and Osteocyte Differentiation.
Untransduced hMSCs as well as CEETnl2- and CEETnl2Is2 transduced hMSC were differentiated to adipocytes and osteocytes as previously described (Anderson, P. et al. CD105 (endoglin)-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities. PLoS ONE (2013)). Briefly, hMSCs were plated in 6-well plates at a density of 20,000 cells/cm2 for adipogenesis, 10,000 cells/cm2 for osteogenesis and incubated in the adipogenic and osteogenic MSCs differentiation BulletKits respectively (Ionza, Basel, Switzerland).
Human ESCs Hematopoietic Differentiation in OP9 Co-Culture System.
Hematopoietic differentiation was induced as described by Ji et al (Ji, J. et al. OP9 stroma augments survival of hematopoietic precursors and progenitors during hematopoietic differentiation from human embryonic stem cells. Stem Cells (2008)). Briefly, the hESCs lines were transferred onto OP9 feeders for 15 days. To evaluate hematopoietic differentiation and eGFP expression at the different days, cells were dissociated with collagenase IV and Tryple (Gibco), resuspended in FACS buffer, filtered through a 70 μm cell strainer (BD Biosciences, Bedford, Mass.) and stained with anti-mouse CD29-FITC (AbD Serotec, Raleigh, GBK), anti-human CD34-PE-Cy7 and anti-human CD45-APC (all from eBiosciences, San Diego, Calif.) and analyzed in a FACS Canto II flow cytometer.
Differentiation of hESCs Towards the Mesenchymal Lineage.
Derivation of MSC-like cell was carried out as previously described (Liu et al., 2012), Briefly, AND-I hESC were treated with 10 μM ROCK for 1 hours, and disaggregated into a single cells with trypsin. The cells were collected in maintenance medium (Embryonic fibroblast conditioned medium supplemented with 4 ng/ml FGF) (Invitrogen), and seeded at a density of 15000/cm2 in Type I collagen coated well tissue culture. 24 hours later, the maintenance medium was supplemented with an equal volume of basal α-MEM (invitrogen) with 10% FBS (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen) and 50 μM magnesium L-ascorbic acid phosphate (Sigma-Aldrich). 10 days later, the medium was replaced with α-MEM (Invitrogen), supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine. The third passage was used for flow cytometry analysis.
FACS Analysis and FACS-Sorting.
Separation of eGFP+hMSCs and hESCs.Dox-induced eGFP+ populations of CEETnl2 and CEETnl2Is2-transduced hMSCs or hESCs were washed and separated using fluorescence-activated cell sorting (FACS) Aria II Flow Cytometer. A MOI=0.4 was used to keep the percentage of transduced hMSCs below 15%. For hESCs a MOI=30 was used to achieve over 30% eGFP+ cells and good expression levels for sorting.
Western Blot
Cells were lysed in RIPA buffer (Sigma) containing protease inhibitors (Sigma Aldrich) at 1×106 cells/100 μl. The lysates (20 μg/sample) were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10% polyacrylamide under reducing conditions) and electrotransferred to nitrocellulose membranes (Bio-Rad Hercules Calif.). Milk-blocked Membranes were probed for 1 hour at room temperature with mouse anti-Tet-repressor (Mobitec; no TET02) at 1:500 dilution at 4° C., and rabbit anti-alpha-tubulin (Santa Cruz, sc-5546). Combination of IRDye 680LT Goat anti-Rabbit IgG (Licors: no 26-68021) and IRDye 800CW Goat anti-Mouse IgG (Licors: no 926-32210) were use at 1:10.000 dilutions to analyzed TetR and tubulin using an Odyssey Image scanner system (LI-COR Biosciences).
Determination of Fold Induction and Leaking Index.
Transduced cells incubated in the presence or absence of Dox (0.1μg/ml) were analyzed by flow cytometry to determine the percentage and Mean Fluorescence Intensity (MFI) of eGFP positive cells as well as the MFI of WLC (WLC=P1 defined as the 7-AAD negative population). The fold induction was calculated using the following formula MFI P1 (+Dox)/MFI P1 (−Dox). Similarly, the Leakiness of the system was determined by using the following formulas [% eGFP+ (−Dox)×100/% eGFP+(+Dox)] or [[% eGFP+ (−Dox)×100/% eGFP+(+Dox)]]×MFI eGFP+(−Dox)). Background (% eGFP+ cells in MOCK transduced cells) were subtracted to the % eGFP+ obtained in the different transductions and conditions. Therefore, when the % eGFP+ (−Dox) is identical in Mock and Transduced cells the leaking is zero and when the % eGFP+ (−Dox) is identical to the % eGFP+(+Dox) the leaking is 100.
DNA Methylation Assay
The promoter methylation was assessed by bisulfite treatment of Genomic DNA and sequencing of the resulting converted gDNA. In the presence of sodium bisulfite, only the unmethylated cytosines are chemically converted to uracil. The genomic DNA was isolated from hESCs at the desired time points using the DNaseasy kit (Qiagen, Crawly, UK). Sodium bisulfite treatment of genomic DNA was performed using the EpiTect bisulfit kit (Qiagene), according to the manufacturer's instruction. The converted gDNA was used for a nested PCR amplification of SFFV and EF1a promoters. The primers used for each amplification is shown in Table 1. The PCR product band was purified and cloned into PCR 2.1 (Invitrogen) according to the manufacturer's instruction. 15-30 clones were randomly selected and sequenced with M13 primers. Identical sequences were eliminated from the study to avoid duplicated clones.
Analysis of the Adipocyte and Osteocyte Differentiation:
The different cells line were incubated in osteogenic and adipogenic MSCs differentiation BulletKit media respectively (Ionza, Basel, Switzerland), and stained with Oil red (Amresco, solon, Ohio) to stain lipid vesicles present in adipocytes and with Alizarin Red S (Sigma) to stain calcium deposits in osteocytes.
mRNA Analysis by RT-qPCR.
Total RNA from hMSCs transduced with CESTln2Is2 or with CEETLN2Is2 was isolated using the Trizol reagent (Invitrogen) and reverse-transcribed using the Superscript first-strand system (Invitrogen). qPCRs were performed using the QuantiTect SYBRGreen PCR kit (Qiagen) on a Stratagene MX3005P system (Agilent Technologies, Santa Clara, Calif.). Q-PCR reactions consisted of 40 cycles at 94° C. (15 sec), then 60° C. (30 sec) and 72° C. (30 Sec). TetR and GADPH specific primers are shown in Table 1. The relative expression was calculated using ΔΔCT method.
Statistical Analysis
All data are represented as mean +/−(SEM). The statistical analysis was performed using the GraphPad Prism software (GraphPad Software, Inc., La Jolla, Calif.) applying the unpaired two-tailed t-test. Statistical significance was defined as a P value<0.05.
Results
In order to generate Tet-On all-in-one LVs that tightly regulate transgene expression in human stem cells we have constructed several LVs harboring different modifications (
Next, we constructed four all-in-one LVs harboring different promoters and insulator combinations (
Expression of the TetRnl2 through the hEF1α promoter in 293T cells decreased the leaking of the vectors in the absence of Dox (Compare CESTnl2 versus CEETnl2 in
In spite of the improvements in leaking and fold induction of the CEETnl2 and CESTnl2 LVs, both vectors still presented high leaking and poor fold induction. Insulators, such as the chicken hypersensitive site 4 (cHS4) (Chung, J. H., et al. A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell (1993)) can shield integrative vectors from nearby regulatory domains favoring a more autonomous regulation and also act as barriers to avoid silencing (Emery, D. W. et al. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci USA (2000); Rivella, S. et al. The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites. J Virol (2000)).
SARs/MARs elements have also been incorporated into retroviral vectors improving their transgene expression and preventing promoter inactivation (Agarwal, M. et al. Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells. J Virol (1998); Dang, Q. et al. Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector. J Virol (2000); Ramezani, A. et al. Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator. Blood (2003)). We have previously constructed the Is2 insulator (combining the HS4-650 and a synthetic SAR element) that was able to enhance expression and avoid silencing of LVs in hESCs (Benabdellah, K., A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells. PLoS ONE (2014)). We hypothesized that the inclusion of this element in the CESTnl2 and CEETnl2 LVs could improve their performance.
As expected, the CESTnl2Is2 LV showed improved expression levels compared to their Is2-negative counterpart (CESTnl2) in the presence of Dox (
Leaking of a regulatable system can be analyzed in different ways. For simplicity, we have used the formula; leaking=[% eGFP+(−Dox)*100/% eGFP+(+Dox) that generate values from 0 (no leaking) to 100 (absence of regulation). However, this analysis does not discriminate between partially-responsive cells from those cells that do not respond to Dox. We have therefore reanalyzed the leaking using an alternative formula; leaking=[% eGFP+ (−Dox)/% eGFP+(−Dox)]×MFI eGFP+cells (−Dox)] to take into account the expression levels of the transduced cells in the absence of Dox (
All together these data indicate that only the CEETnl2Is2 LV was able to regulate transgene expression in 293T harboring one vector copy number per cell (vcn/c). As a down side, we detected a reduction on the titer (2-3 fold) of the Is2-LVs compared to their Is2-negative counterparts (data not shown).
The fold induction parameter indicates the increment of eGFP expression of the total population upon the addition of Dox (see M&M for details). Since we kept transduction below 40% (to obtain cell populations harboring only one vcn/c), the fold induction is underestimated. To study the potency of the system we further analyzed the inducibility of the Lent-On-Plus LVs (CEETnl2 and CEETnl2Is2) on cell populations that are 100% responsive to Dox (
We next tested the new inducible systems in hMSCs. We transduce hMSCs with the CESTnl2, CESTnl2Is2, CEETnl2 and CEETnl2Is2 LVs at a MOI=1 and 10 days later analyzed for GFP expression in the presence or absence of Dox (
Inducible systems should be able to maintain transgene regulation upon cellular expansion and/or differentiation. We therefore transduced hMSCs with the CEETnl2 and CEETnl2Is2 LVs and studied transgene regulation after expansion (
Finally we aimed to generate pure populations of CEETnl2 and CEETnl2Is2-cells harboring 1 vcn/c. hMSCs were transduced at low MOI to achieve 9-15% eGFP+ cells upon induction with Dox. Two weeks later eGFP+ hMSCs were sorted to a purity of 99% (
The generation of pluripotent stem cells expressing the desired transgene under the tight control of an inducer has been a very difficult task to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the side effects of the transactivators. We hypothesized that the use of insulated LVs expressing the TetRnl2 through stable promoters could overcome previous existing limitations to generate Dox-inducible hESCs. To investigate this possibility, we first tested the Dox-responsiveness of two hESCs lines (AND-1 and H9) transduced with the CESTnl2, CESTnl2Is2, CEETnl2 and CEETnl2Is2 LVs at MOI=5 (
We next generated sorted CEETnl2- and CEETnl2Is2-transduced hESCs in order to study Dox responsiveness in a purer population. hESCs were transduced at MOI=30 and two weeks later, CEETnl2- and CEETnl2Is2-transduced hESCs were sorted to a purity of 74 and 50% respectively (
Transgene silencing due to promoter methylation of integrative RVs has been a major problem for stable expression in hESCs. Several studies have shown that pluripotent cells can block expression of exogenous retroviral elements by complex defense mechanisms (Cherry, S. R. et al. Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol Cell Biol (2000)). It has been previously described that the SFFV promoter is silenced through methylation in human pluripotent cells (Herbst, F. et al. Extensive methylation of promoter sequences silences lentiviral transgene expression during stem cell differentiation in vivo. Mol Ther (2012)) while the hEF1α promoter is highly resistant allowing stable transgene expression in these cell types (Hong, S. et al. Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells. Mol Ther (2007); Wang, R. et al. Promoter-dependent EGFP expression during embryonic stem cell propagation and differentiation. Stem Cells Dev (2008); Xia, X. et al. Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner. Stem Cells Dev (2007)). We therefore analyzed the methylation profiles of the SFFV and hEF1α promoters in CESTnl2- and in CEETnl2-transduced hESCs (
Ideally, the inducible systems for pluripotent stem cells must be also able to maintain drug responses upon differentiation toward different cell types. We then analyzed whether the Lent-On-Plus systems (CEETnl2 and CEETnl2Is2) maintained the Dox responsiveness in mesenchymal and hematopoietic cells derived from transduced hESCs (
hESCs transduced with the CEETnl2 and CEETnl2Is2 LVs at high MOI (vcn/c of 2.5 and 1.9 respectively) (
We finally differentiated CEETnl2- and CEETnl2Is2-transduced hESCs toward the hematopoietic lineage using the OP9 differentiation protocol as previously described33 (see M&M for details). The cells were maintained in the presence or absence of Dox from day 1 of differentiation and the eGFP expression was analyzed in the different hematopoietic populations at day 8 and day 15 (
| Number | Date | Country | Kind |
|---|---|---|---|
| P201631406 | Nov 2016 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/078246 | 11/3/2017 | WO | 00 |
