Neurotrophic factor and optogenetic system based lentiviral system for use in retinal diseases

Abstract

In the invention, an HIV or EIAV-based lentiviral vector system to be used in the treatment of retinal diseases by transdifferentiating mesenchymal stem cells isolated from adipose tissue, bone marrow or umbilical cord tissue into photoreceptor cells using neurotrophic factors through optogenetics, and a method for creating this system is described. In this system, pHIV1 and pEV53D vectors are individually packaged with the neurotrophic factors BDNF, CNTF, PEDF, NTF-3 and the promoters CMV, RHO, hGRK, hRP1, and the optogenetic tool Opto-mGLUR6.

Description

[0001] NEUROTROPHIC FACTOR AND OPTOGENETIC SYSTEM BASED LENTIVIRAL SYSTEM FOR USE IN RETINAL DISEASES

[0002] Technical Field

[0003] The invention relates to an HIV or ElAV-based lentiviral vector system to be used in the treatment of retinal diseases by transdifferentiating mesenchymal stem cells isolated from adipose tissue or bone marrow tissue into photoreceptor cells using neurotrophic factors through optogenetics, and to a method for creating this system. By means of the photoreceptor cells created by cell transdifferentiation with the lentiviral vector system of the invention, pre-clinical studies of genetic diseases occurring in photoreceptor cells can be carried out effectively.

[0004] State of the Art

[0005] Stem cells are cells that have the ability to differentiate and self-renew along different lineages and form the structure of all tissues and organs of the body. Stem cells can also perform critical roles such as immune regulation and homoeostasis. These cells are divided into two main categories, as defined in the literature: pluripotent (embryonic stem cells and induced pluripotent stem cells) and non-embryonic or somatic stem cells (adult stem cells). Pluripotent stem cells have the ability to differentiate into all cells in the adult body, but adult stem cells are specific to a tissue or organ and differentiate to give specialised cell types of that tissue or organ [1], Embryonic stem cells are obtained from the inner cell mass of the blastocyst, and since the extraction process requires the destruction of the human embryo, it is unethical to use these cell types in research because it may have potential consequences that may lead to human rights violations [2], Therefore, induced pluripotent and adult mesenchymal stem cells (MSCs) are frequently used in experimental and clinical studies. Induced pluripotent stem cells are derived from skin or blood cells that have been reprogrammed into an embryonic-like pluripotent state, enabling the development of an unlimited source of any type of human cell needed for therapeutic purposes. Mesenchymal stem cells (MSG) are stromal cells that have the ability to self-renew and also exhibit multilineage differentiation and can be isolated from various tissues such as umbilical cord, endometrial polyps, menstrual blood, bone marrow, adipose tissue, etc. Unlike induced pluripotent stem cells, more effective results are obtained in clinical applications with the use of mesenchymal stem cells in gene manipulations.

[0006] Mesenchymal stem cells are especially important in terms of cellular therapies in regenerative medicine, both in pre-clinical animal studies and at the clinical level, by means of their ability to transform into different cell types [3]. Mesenchymal stem cells cells can be obtained from very different tissues such as placenta, adipose tissue, blood, bone marrow, dermis, Wharton's gel (mucosal connective tissue found in the umbilical cord) and foreskin. MSCs obtained from many different tissues are characterised by endoderm, ectoderm and mesoderm differentiation such as bone, liver, neuron or retina cells under in vitro conditions, and this differentiation is carried out by transcription factors, growth factors and induction chemicals. In order to ensure differentiation, Mesenchymal stem cells factors are packaged by viruses and given to the cell that is desired to differentiate. The aim is to induce differentiation by ensuring the expression of these factors within the cell. Vectors used for this purpose are generally adeno-associated viruses, retroviral, adenoviral and lentiviral vectors. Lentiviral vectors are frequently preferred in gene therapies because they have a large genetic capacity and the ability to transform both dividing and non-dividing cells. In general, neurotrophic factors, which are secretomes secreted from MSCs, prevent differentiation, cell survival and triggering of apoptosis. For this reason, neurotrophic factors are widely used, especially in stem cell treatment studies. Neurotrophic factors activate the P13 / KAKT, MAPK / ERK, PLC / IP3 / PKC and JAK / STAT3 signalling pathways within the cell, especially preventing the cell from entering apoptosis and thus cell loss. Additionally, neurotrophic factors play an important role in neuronal target interactions during the embryonic and adult periods, and cells proliferate in response to these factors. In this way, neurotrophic factors regulate the proliferation and terminal differentiation of stem cells [4],

[0007] Retinal cells, one of the cell types into which mesenchymal stem cells can differentiate, consist of specialised cells that convert light into meaningful signals for the brain. Photoreceptor cells, called rod and cone, are a special type of neuroepithelial cell found in the retina and capable of visual phototransduction, and they play an important role in providing colour vision and night vision. MSC differentiation and transplantation is a widely used approach in retinal diseases, especially in the treatment of different retinopathic diseases such as Stargardt's disease, diabetic retinopathy, glaucoma and age-related macular degeneration, which cause apoptosis of photoreceptor cells and result in vision loss [5]. However, the rapid degradation of stem cells and the inability of cells to differentiate in numbers that would eliminate or reduce the pathophysiology of these diseases limit the effectiveness of these approaches. Additionally, optogenetic tools are also used in retinal diseases. Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the cell-specific metabotropic glutamate receptor mGluR6 and the light-sensitive domains of melanopsin, is an optogenetic tool that has been shown to reliably rescue vision at the retinal, cortical and behavioural levels under moderate daylight illumination [6] These studies show that promising new "optogenetic" treatments under pre-clinical evaluation use a modified virus to introduce light-sensitive proteins into surviving retinal cells, turning them into "replacement photoreceptors" and thus restoring vision. Additionally, they offer an effective therapeutic approach by increasing transcription activation in cells and keeping it at a stable level. However, there are many factors that limit the feasibility of optogenetic therapy. For example, existing photosensitive proteins respond only to unnaturally high light intensities and use foreign signalling mechanisms to activate target retinal cells. This situation depends on more than one variable, depending on the power and electrical capacity of the optogenetic tools, laser or light system used, so deficiency in any of them leads to negative effects.

[0008] A study conducted by Najafabadi et al. in the state of the art relates to an investigation of the effectiveness of blue light stimulation on the neuroretinal differentiation of Opto- mGluR6 engineered mouse retinal pigment epithelium (mRPE) and bone marrow mesenchymal stem cells (BMSCs). mRPE and BMSCs used in Mesenchymal stem cells study are selected for the optogenetic study due to their ability to differentiate into retinaspecific neurons, and mRPE cells and BMSCs were stimulated with blue light for 5 days after being transduced with the AAV-MCS-IRES-EGFP-Opto-mGluR6 viral vector. The plasmid containing said viral vector is adeno-associated virus (AAV). Since AAVs are not integrated into the genome, they cannot be a method for permanent treatment at once. As a result of the study, retina-specific neuron differentiation is observed in mRPE cells and in BMSCs. However, this differentiation occurs in the mouse retinal pigment epithelium cell type, so it cannot be used for therapeutic purposes in humans. In addition, the Opto-mGluR6 designed in the study only provides the effectiveness of blue light stimulation, so the number of target cell types is limited. Additionally, adeno-associated virus has been used as a method of viral delivery, but during the use of this virus in neuron differentiation, a decrease in expression may occur over time due to episomal loss through degradation, small packaging capacity, low titers, and a strong cell-mediated immune response.

[0009] A study conducted by Miyoshi et al. in the state of the art concerns an HIV-based lentiviral vector system that can in vivo transduce terminally differentiated neurons of the brain. In this study, the ability of HIV vectors to transfer genes to retina cells is evaluated. It is mentioned that the GFP gene under the control of the cytomegalovirus promoter is efficiently expressed in both photoreceptor cells and retinal pigment epithelium, whereas the use of the rhodopsin promoter results in expression predominantly in photoreceptor cells. The study demonstrates that HIV-based vectors can efficiently transfer and express a transgene in retinal cells, specifically RPE and photoreceptor cells. The study includes that HIV-based systems can be used successfully in gene transfer, especially in photoreceptor and Retinal Pigment Epithelium (RPE)-related retinal disorders. However, the transdifferentiation of mesenchymal stem cells into photoreceptor cells is not mentioned with this vector system. In addition, the reason for using CMV and RHO as promoters in the study is to specifically target the retinal cells present in the study, but with the use of mesenchymal stem cells that are not specifically differentiated into a cell type, it is not possible for these cells to differentiate into photoreceptor cells. In addition, activation of cells' signalling pathways by light of certain wavelengths by Opto-mGluR6 is not noted in the study.

[0010] Patent numbered LIS2021 155663A1 in the state of the art relates to a recombinant lentiviral vector containing a gene encoding a brain-derived neurotrophic factor (BDNF) protein and a cell transfected with a lentivirus prepared using the vector. In the invention, it is mentioned that the recombinant lentiviral vector can regulate gene expression by a promoter and this promoter can be a cytomegalovirus (CMV), respiratory syncytial virus (RSV), human elongation factor-1 alpha (EF-1a) or tetracycline response elements (TRE), and also that the host cell may be a human embryonic stem cell (hES), a bone marrow stem cell (BMSC), a mesenchymal stem cell (MSC), a human neural stem cell (hNSC), a limbal stem cell or an oral mucosal epithelial cell. In the invention, there is no system or element that will trigger the activity of BDNF expression, therefore it is neither possible to ensure proliferation of the cells nor to ensure differentiation into photoreceptor cells.

[0011] Due to reasons such as the limitations and inadequacies of the solutions in the state of the art and the fact that cell loss in cell transdifferentiation applications in the state of the art causes a decrease in the effectiveness level of therapy, rapid degradation of stem cells, inability to differentiate in a number of cells that would eliminate or reduce the pathophysiology of diseases, the cells differentiated in the state of the are not specific to human cell lines, and therefore there is no person-specific therapeutic approach that can be clinically returned to the person in the ex vivo environment, it has become necessary to make an improvement in lentiviral vector systems.

[0012] Brief Description and Aims of the Invention

[0013] In the invention, an HIV or ElAV-based lentiviral vector system to be used in the treatment of retinal diseases by transdifferentiating mesenchymal stem cells isolated from adipose tissue, bone marrow or umbilical cord tissue into photoreceptor cells using neurotrophic factors through optogenetics, and a method for creating this system is described. In this system, pHIV1 and pEV53D vectors are packaged with neurotrophic factors (BDNF, CNTF, PEDF and NTF-3) and promoters (CMV, RHO, hGRK and hRP1 ) and the optogenetic tool Opto-mGLUR6. The lentivirus system prepared to induce mesenchymal stem cell (MSC) differentiation is transferred to isolated MSC cells under in vitro conditions. In this way, it is possible to transfer functional synthetic neurotrophic genes and deliver the optogenetic tool. Cells that undergo neurotrophic gene transfer and optogenetic tool transfer are exposed to light at the appropriate wavelength in order for Opto-mGLUR6 to perform its function. With the expression of transmitted factors and Opto-mGLUR6, bone marrow and adipose-derived MSCs differentiate into photoreceptor cells. Differentiated cells are returned to the isolated individual in an ex vivo environment and used in the treatment of retinal diseases such as Stargardt's disease, diabetic retinopathy, glaucoma and age-related macular degeneration.

[0014] The aim of the invention is to provide an HIV or ElAV-based lentiviral vector system for use in the effective treatment of retinal diseases. The system ensures the differentiation of a number of cells that will eliminate or reduce the pathophysiology of retinal diseases. In the lentiviral vector system that is the subject of the invention, there are two different plasmids. In this way, not only photoreceptor cell transformation is increased through different promoter sequences and neurotrophic factors, but also by using two different vectors to carry these sequences, the use of the most optimal vector among the vectors is guaranteed and an effective treatment is provided.

[0015] An aim of the invention is to provide a personalised therapeutic approach in retinal diseases. In the invention, after the mesenchymal stem cells taken from the person are differentiated into photoreceptor cells by means of the HIV or ElAV-based lentiviral vector system, the differentiated cells are clinically returned to the same person and the functionality of healthy photoreceptor cells is demonstrated in the person.

[0016] An aim of the invention is to provide an HIV or ElAV-based lentiviral vector system which enables increased therapeutic effect by preventing cell loss during cell transdifferentiation and a method for creating this system. In the therapeutic approach, which is the main subject of the invention, the gene-induced deteriorations experienced in retinal genetic diseases are prevented by lentiviral transfer and the loss of specific photoreceptor cells that play a role in vision by differentiating MSG cells.

[0017] Detailed Description of the Invention

[0018] In the invention, an HIV or ElAV-based lentiviral vector system to be used in the treatment of retinal diseases by transdifferentiating mesenchymal stem cells isolated from adipose tissue, bone marrow or umbilical cord tissue into photoreceptor cells using neurotrophic factors through optogenetics, and a method for creating this system is described. In the presence of two agents, the light system as a physical agent and the neurotrophic factors as a chemical agent, transcription factors that can be kept under control by the chemical system in the system are connected to the promoter regions and the transformation of mesenchymal stem cells into photoreceptor cells is achieved. In the invention, BDNF, CNTF, PEDF and NTF-3 as transcription factors, CMV, RHO, hGRK and hRP1 as promoters and Opto-mGLUR6 as optogenetic tool are individually packaged with pHIV1 and pEV53D, which are HIV and ElAV-based lentiviral vectors, respectively.

[0019] Method of creating an HIV or ElAV-based lentiviral vector system to ensure the transdifferentiation of mesenchymal stem cells into photoreceptor cells comprises the process steps of: i. Creating SEQ ID NO:1 -4, SEQ ID NO:6-9, SEQ ID NO:11 -14, SEQ ID NO:16-19 sequences as a result of combining promoters with neurotrophic factors, ii. Creating SEQ ID NO:5, SEQ ID NQ:10, SEQ ID NO:15 and SEQ ID NQ:20 sequences as a result of combining promoters with an optogenetic tool, ill. packaging of promoter / neurotrophic factor sequences combined with lentiviruses in step (i), and iv. packaging of promoter / optogenetic tool sequences combined with lentiviruses in step (ii).

[0020] In the invention, CMV, RHO, hGRK and hRP1 promoters are each associated with BDNF, CNTF, PEDF and NTF-3, respectively. By combining CMV and BDNF, CMV-BDNF having the nucleotide sequence SEQ ID NO:1 is obtained. By combining CMV and CNTF, CMV- CNTF having the nucleotide sequence SEQ ID NO:2 is obtained. By combining CMV and PEDF, CMV-PEDF having the nucleotide sequence SEQ ID NO:3 is obtained. By combining CMV and NTF-3, CMV-NTF3 having the nucleotide sequence SEQ ID NO:4 is obtained. By combining RHO and BDNF, RHO-BDNF having the nucleotide sequence SEQ ID NO:6 is obtained. By combining RHO and CNTF, RHO-CNTF having the nucleotide sequence SEQ ID NO:7 is obtained. By combining RHO and PEDF, RHO- PEDF having the nucleotide sequence SEQ ID NO:8 is obtained. By combining RHO and NTF-3, RHO-NTF-3 having the nucleotide sequence SEQ ID NO:9 is obtained. By combining hGRK with BDNF, hGRK-BDNF with the nucleotide sequence SEQ ID NO: 11 is obtained. By combining hGRK and CNTF, hGRK-CNTF with the nucleotide sequence SEQ ID NO: 12 is obtained. By combining hGRK with PEDF, hGRK-PEDF with the nucleotide sequence SEQ ID NO: 13 is obtained. By combining hGRK and NTF-3, hGRK- NTF-3 with the nucleotide sequence SEQ ID NO: 14 is obtained. By combining hRP1 and BDNF, hRP1 -BDNF having the nucleotide sequence SEQ ID NO: 16 is obtained. By combining hRP1 and CNTF, hRP1 -CNTF having the nucleotide sequence SEQ ID NO: 17 is obtained. By combining hRP1 and PEDF, hRP1 -PEDF having the nucleotide sequence SEQ ID NO: 18 is obtained. By combining hRP1 and NTF-3, hRP1 -NTF- 3having the nucleotide sequence SEQ ID NO: 19 is obtained. After the obtained promoter / neurotrophic factor sequences, CMV-Opto-mGLUR6 sequence having the nucleotide sequence SEQ ID NO: 5 is obtained by combining CMV with Opto-mGLUR6, RHO-Opto-mGLUR6 sequence having the nucleotide sequence of SEQ ID NQ:10 by combining RHO with Opto-mGLUR6, hGRK-Opto-mGLUR6 sequence having the nucleotide sequence of SEQ ID NO:15 by combining hGRK with Opto-mGLUR6, hRP1 - Opto-mGLUR6 sequence having the nucleotide sequence of SEQ ID NO: 20 by combining hRP1 with Opto-mGLUR6, and promoter / optogenetic tool sequences are obtained. These sequences are integrated into the genome and prevent cells that are mutant or whose gene does not work, from dying and making false differentiation. After the sequences are obtained, the promoter-neurotrophic factor / optogenetic tool sequences with the nucleotide sequences of SEQ ID NO: 1 -20 are packaged with the pHIV1 plasmid (the HIV lentiviral vector it encodes) and the pEV53D plasmid (the EIAV lentiviral vector it encodes). For this packaging process, plasmids with BDNF, CTNF, PEDF, NTF3 and mGLUR6 coding sequences are synthesised. Then, factor-encoding plasmid DNAs are transformed into E. coli DH5 strain and the plasmids are propagated. A purification kit is used to ensure that the propagated plasmids do not contain endotoxin. Plasmids with BDNF, CNTF, PEDF, NTF3 and mGLUR6 coding sequences are treated with polyethyleneimine to condense DNA and then packaged in lentivirus using HEK293-T (human kidney cell line) as host cells. As a result of this packaging, pHIV1 vectors expressing SEQ ID NO:1 -20 sequences and pEV53D vectors expressing SEQ ID NO:1 - 20 sequences are obtained. (Culture content: DMEM with 10% Fetal bovine serum and 1 % penicillin / streptomycin L-Glutamine medium). Packaged recombinant lentiviruses are harvested from the supernatant of HEK293-T cell cultures at 48 hours after transfection. After completion of the lentivirus titration test and other quality control tests, including sterility and purity, pHIV1 plasmids encoding any of the nucleotide sequences of SEQ ID NO: 1 -20 and pEV53D plasmids encoding any of the nucleotide sequences of SEQ ID NO: 1 -20 are created. The HIV or ElAV-based lentiviral vector system of the invention also comprises the pHIV1 or pEV53D plasmid encoding any of the mentioned SEQ ID NO: 1 -20 or any combination of these plasmids. The lentiviral vector system is stored at - 80°C. In addition, in this HIV or ElAV-based lentivirus system, 1 x 106or higher viral particles per millilitre are obtained by performing virus titration by performing flow cytometry on Jurkat cells expressing EGFP.

[0021] After obtaining the lentiviral vector system of the invention, in order to ensure the transdifferentiation of mesenchymal cells isolated from adipose tissue, bone marrow or umbilical cord tissue into photoreceptor cells, the supernatant is collected and purified after cell culture after recombinant lentiviruses are transfected into HEK293-T cells. By counting the Jurkat cell line kept in culture, 10,000 cells are prepared in 100 ml of complete medium (Culture content: RPMI (growth medium) with glutamine and HEPES (zwitterionic sulfonic acid buffer), 10% fetal bovine serum, 1% Penicillin / streptomycin antibiotic mixture, 1% non-essential amino acid, 1% sodium pyruvate, 1% vitamin). Jurkat cells in 100 pl of medium are seeded in 96-well plates. The wells are adjusted to have 10 ml, 3 ml, 1 ml, 0.3 ml, 0.1 ml and 0.03 ml of 100X concentrated lentivirus solution encoding BDNF, CNTF, PEDF, NTF3 and mGLUR6 per 50 ml. In the evaluation of photoreceptor cells differentiated from mesenchymal stem cells, differentiation is detected and confirmed by measuring the expression levels of RHO, CRX, Recoverin, TBP, PKC, Peripherin and OTX2 photoreceptor marker genes. REFERENCES

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Claims

CLAIMS1. An HIV or ElAV-based lent! viral vector system, comprising• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:1 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:2 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:3 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:4 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:5 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:6 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:7 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:8 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:9 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:10 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:11 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:12 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:13 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:14 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:15 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:16 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:17 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:18 sequence,• pHIV1 vector or pEV53D vector encoding the SEQ ID NO:19 sequence, and• pHIV1 vector or pEV53D vector encoding the SEQ ID NQ:20, or any combination thereof.

2. An HIV- or ElAV-based lentiviral vector system according to claim 1 for transdifferentiating mesenchymal stem cells into photoreceptor cells.

3. An HIV- or ElAV-based lentiviral vector system according to claim 1 or 2 for use in the treatment of retinal diseases.

4. Method of creating an HIV or ElAV-based lentiviral vector system to ensure the transdifferentiation of mesenchymal stem cells into photoreceptor cells comprises the process steps of: i. creating SEQ ID NO:1 -4, SEQ ID NO:6-9, SEQ ID NO:11 -14, SEQ ID NO:16-19 sequences as a result of combining promoters with neurotrophic factors, ii. creating SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15 and SEQ ID NQ:20 sequences as a result of combining promoters with an optogenetic tool, iii. packaging of SEQ ID NO:1 -4, SEQ ID NO:6-9, SEQ ID NO:11 -14, SEQ ID NO:16-19 promoter / neurotrophic factor sequences combined with lentiviruses in the process step (i), and iv. packaging of SEQ ID NO:5, SEQ ID NQ:10, SEQ ID NO:15 and SEQ ID NQ:20 promoter / optogenetic tool sequences combined with lentiviruses in the process step (ii).

5. A method according to claim 4, wherein the promoter mentioned in step (i) or (ii) is CMV, CMV, RHO, hGRK or hRP1 .

6. A method according to claim 4, wherein the neurotrophic factor mentioned in the process step (i) is BDNF, CNTF, PEDF or NTF-3.

7. A method according to claim 4, wherein the optogenetic tool mentioned in the process step (ii) is Opto-mGLUR6.

8. A method according to claim 4, wherein the lentivirus mentioned in step (iii) or (iv) is pHIV1 or pEV53D.

9. HIV-based lentiviral vector system created by a method according to claim 4 for use in the treatment of retinal diseases.