Hybrid peptide for gene transfer

Abstract

The invention relates to a hybrid peptide consisting of amino acid sequences that bind nucleic acids on one side and cell membrane on the other side, in the fields of biology, genetics and chemistry. The invention particularly relates to a hybrid peptide developed to ensure the transfer of genes into the cell in the treatment of genetic diseases and incurable diseases such as some types of cancer.

Description

[0001] HYBRID PEPTIDE FOR GENE TRANSFER

[0002] Technical Field

[0003] The invention relates to a hybrid peptide, consisting of amino acid sequences that bind nucleic acids on one side and the cell on the other side, in the fields of biology, genetics and chemistry.

[0004] The invention particularly relates to a hybrid peptide developed to enable the transfer of genes into cells, in the treatment of genetic diseases and incurable diseases such as some types of cancer.

[0005] State of Art

[0006] There are many genetic diseases that affect humans. In order to treat these diseases, an important goal is to transfer the gene carrying the correct DNA sequence into cells instead of the faulty gene that causes the disease. Gene therapies that directly target cancer cells or genetic modifications that enable immune system cells to target cancer cells are becoming increasingly important in the treatment of diseases such as many types of cancer that cannot be treated with current methods [1]. Cancer vaccines that have emerged in recent years or vaccines developed against Covid-19 perform their functions by transferring RNA molecules into the cells and ensuring that antigens that will stimulate the immune system are produced in the cells [2],

[0007] Today, gene therapy is gaining increasing importance for the treatment of genetic diseases and incurable diseases such as some types of cancer. Gene therapy is performed by introducing a gene into cells from outside. The most important problem encountered in gene therapy is the transfer of the desired gene into the cell. Various gene carrier vectors are used for gene transfer. Viruses, which are the most prominent among these, are generally obtained by removing some genes that can cause disease and adding genes that are planned to be put into cells instead. There is a discussion about whether viruses are safe or not, as there are concerns about the changes they can undergo in the human body and whether they can be a factor that causes infection or cause cancer [3]. Liposomes obtained by packaging nucleic acids with various lipids are thought to be more reliable vectors [4], RNA vaccines, which have been widely and effectively used in recent years, have been packaged with such lipids and sent into the cell, successfully producing the desired antigen from mRNA and ensuring the formation of immunity against it. During the Covid-19 pandemic, mRNA vaccines prepared in this way were administered to millions of people. Despite all this, the lipids used in gene transfer are very difficult and expensive to produce. In addition, myocarditis (inflammation of the heart muscle) has occurred in individuals who received mRNA vaccines made with these lipids, although this is not very common [5]. In order to eliminate these problems in the known state of the art, an easy-to-produce and inexpensive method is needed.

[0008] As a result of the research carried out in the literature, application numbered DE4005152A1 was encountered. The said document relates to the transformation of plant protoplasts with DNA-histone complex - providing high and reproducible transfer rate and expression of foreign genes. In the said document, peptide sequences belonging to histone proteins were used to transfer DNA molecules into the cell. Histone proteins have the ability to bind to DNA. Since they are generally rich in positive charges, they neutralize the negative charges of DNA, making it easier for it to approach the cell membrane. However, since they do not carry a peptide sequence related to attaching to and passing through the cell membrane, they cannot effectively provide DNA entry into the cell on their own.

[0009] Another application, numbered CN107794280A, relates to a peptide gene carrier that penetrates into the target cell and its application. In the said document, only cellpenetrating peptides were used to deliver DNA into the cell. For this reason, they cannot effectively ensure the entry of DNA into the cell independently. However, the document does not contain any information specifically about adding amino acid sequences of histone proteins to DNA in order to ensure more efficient transfer of the gene to the cell.

[0010] The document numbered CA2189975A1 , which was found as a result of the research carried out in the literature, relates to a method for high-efficiency, stable integration of DNA into a eukaryotic genome. The said document aims to facilitate its transport to the nuclear genome by forming a complex with the DNA molecule with the help of a synthetic polypeptide containing a nuclear localization signal. However, the document only uses peptide sequences that bind to nucleic acids and does not mention the use of peptide sequences that enable it to pass through the cell membrane more easily and effectively.

[0011] The patent application numbered CN103173491 A describes the application of a fusion protein NLS-I-Scel (Nuclear Localization Sequence-l-endonuclease) containing a nuclear localization sequence in the preparation of a reagent that mediates mammalian gene transfer. However, this document does not contain any information about the hybrid peptide, which contains peptide sequences that specifically bind to nucleic acid and enable entry into the cell.

[0012] As a result, due to the negativities described above and the inadequacy of existing solutions on the subject, it has become necessary to make a development in the relevant technical field.

[0013] Object of the Invention

[0014] The invention is inspired by the current situation and aims to solve the above-mentioned negativities.

[0015] The main object of the invention is a hybrid peptide consisting of cell penetrating peptide and histone protein sequences that can bind to the cell membrane and enter the cell at one end and bind to DNA at the other end, eliminating the existing problems in transferring the gene into the cell. When this peptide is mixed with nucleic acids, it binds to them by histone sequences and surrounds them, leaving the parts that will bind to the cell membrane outside. When this formation, which we also call “peptosome”, encounters a cell, it allows the membrane to wrap itself with the parts that attach to the cell membrane and allow the nucleic acid to enter the cell. Histone sequences also carry a nuclear localization signal, enabling their transport to the nucleus. If the inserted nucleic acid is in the DNA structure, mRNA production begins from the genes it carries, which then passes into the cytoplasm and initiates protein synthesis.

[0016] The object of the invention is to provide a safe product that can be used to transfer the gene carrying the correct DNA sequence into cells instead of the faulty gene that causes the disease for the treatment of diseases.

[0017] An object of the invention is to provide a product that is easy and inexpensive to manufacture.

[0018] In order to achieve the objects described above, the invention is a hybrid peptide, which comprises peptide sequences that attach to the cell membrane and provide entry into the cell at one end, and peptide sequences that bind to nucleic acids and exhibit nuclear localization signal properties at the other end. The structural and characteristic features of the invention and all its advantages will be understood more clearly by means of the detailed description given below, and therefore the evaluation needs to be made by taking this detailed description into consideration.

[0019] Detailed Description of the Invention

[0020] In this detailed description, the product which is the subject of the invention and its preferred embodiments are described only for the purpose of better understanding the subject.

[0021] The present invention is a hybrid peptide, comprising peptide sequences that attach to the cell membrane and provide entry into the cell at one end, and peptide sequences that bind to nucleic acids and exhibit nuclear localization signal properties at the other end.

[0022] Peptide means a small protein. V=valine; G=Glycine; K=Lysine; A=Alanine; R=Arginine and H=Histidine.

[0023] The mentioned peptide sequences consist of the VGVGKGGAKRHR amino acids.

[0024] The peptide sequences of the hybrid peptide of the invention, which bind to the said nucleic acids and exhibit nuclear localization signal properties, consist of the GKGGAKRHR portion of the histone 4 protein. The sequence mentioned here is a sequence of 9 amino acids from the 12th to the 20th amino acid of the histone 4 protein.

[0025] The hybrid peptide of the invention enables easy insertion of nucleic acids into the cell.

[0026] This invention, which is easy to produce and inexpensive, was developed to eliminate the problems in the known state of the art. The invention is a hybrid peptide consisting of histone protein sequences, one end of which can bind to the cell membrane and enter, and the other end of which binds to DNA. When the hybrid peptide, which is the subject of the invention, is mixed with nucleic acids, it binds to them with the histone sequences and wraps them, while the parts that will bind to the cell membrane remain outside. When the hybrid peptide (this formation we call "Peptosome") encounters a cell, it enables the membrane to wrap itself with its parts that attach to the cell membrane and the nucleic acid to enter the cell. Histone sequences also carry a nuclear localization signal, enabling their transport to the nucleus. If the inserted nucleic acid is in the DNA structure, mRNA production begins from the genes it carries, which then passes into the cytoplasm and initiates protein synthesis.

[0027] Experimental studies and results

[0028] In order to find the hybrid peptide that will best introduce nucleic acids into the cell, peptides consisting of the cell-entering peptide and the DNA-binding region of the histone 4 protein shown in Table 1 below were synthesized. A plasmid carrying the green fluorescent protein gene was packaged with these and inserted into HeLa cells. The cells were examined under a fluorescent microscope and the appearance of light showed that the plasmid had entered the cell and green fluorescent protein was produced. Among the hybrid peptides in Table 1 , the most effective gene transport hybrid peptide was determined to be TM3, which consists of the amino acid sequence VGVGKGGAKRHR. The hybrid peptide, which is the subject of the invention, consists of the amino acids VGVGKGGAKRHR, which are peptide sequences that attach to the cell membrane and allow entry into the cell.

[0029] Table 1 Hybrid peptides

[0030] TM peptides Sequences

[0031] TM1 VTVLAGALAGVGVGKGGAKRHR

[0032] TM2 VTVLAGGKGGAKRHR

[0033] TM3 VGVGKGGAKRHR

[0034] TM4 GKGGAKRHR

[0035] TM5 VTVLAGALAGVGV

[0036] TM6 AAVLLPVLLAAPGKGGAKRHR

[0037] TM7 LGLGKGGAKRHR

[0038] TM8 LGVGKGGAKRHR

[0039] In the study carried out to investigate the effectiveness of hybrid peptides in introducing DNA into the cell, pcDNA 3-EGFP plasmid was used, whose entry into the cell can be monitored by means of the green fluorescent protein it produces when it enters the cell. Plasmids purified after being produced in E. coliwere mixed with the hybrid peptides of the invention at different ratios and added onto HeLa cells grown in cell culture medium. Cells were incubated at 370 in 5% CO 2; after 24 and 48 h, cells were evaluated for fluorescence under a fluorescence microscope. It was determined what percentage of all cells emitted light, that is, internalized the plasmid and produced green fluorescent protein. The transformation efficiency of each hybrid peptide (peptosome) of the invention was compared with "Lipofectamine 2000", which is currently in widespread use and used in liposome preparation. It was determined that the most effective transforming peptide was TM3. “Lipofectamine 2000” transformed 27% and 52% of the cells at 24 and 48 hours, while this rate was 52% and 76% for TM3, respectively.

[0040] References

[0041] 1. Wang, D., Tai, P. W. L., & Gao, G. (2019). Adeno-associated virus vector as a platform for gene therapy delivery. Nature reviews. Drug discovery, 18(5), 358- 378. https: / / doi.Org / 10.1038 / S41573-019-0012-9 2. Ndwandwe, D., & Wiysonge, C. S. (2021 ). COVID-19 vaccines. Current opinion in immunology, 71 , 1 11 -116., Saldanha, L., & Vale, N. (2022). The First Approved COVID-19 Vaccines: The Road to Cancer Vaccines. International Journal of Translational Medicine, 2(3), 309-331.

[0042] 3. Cevher, E., Demir, A., & Sefik, E. (2012). Gene Delivery Systems: Recent Progress in Viral and Non-Viral Therapy. InTech, doi 10.5772 / 53392).

[0043] 4. Ramamoorth, M., & Narvekar, A. (2015). Non-viral vectors in gene therapy- an overview. Journal of clinical and diagnostic research: JCDR, 9(1 ), GE01-GE6. https: / / doi.Org / 10.7860 / JCDR / 2015 / 10443.5394

[0044] 5. Bozkurt, B., Kamat, I., & Hotez, P. J. (2021 ). Myocarditis with COVID-19 mRNA vaccines. Circulation, 144(6), 471 -484.

Claims

CLAIMS1. A hybrid peptide, characterized by comprising; peptide sequences that attach to the cell membrane and provide entry into the cell at one end and peptide sequences that bind to nucleic acids and exhibit nuclear localization signal properties at the other end.

2. The hybrid peptide according to claim 1 , characterized in that; the peptide sequences consist of the amino acids VGVGKGGAKRHR.

3. The hybrid peptide according to claim 1 , characterized in that; peptide sequences that bind to nucleic acids and exhibit nuclear localization signal properties consist of the GKGGAKRHR portion of the histone 4 protein.

4. The hybrid peptide according to claim 3, characterized in that; the sequence mentioned is a sequence consisting of 9 amino acids from the 12th amino acid to the 20th amino acid.

5. The hybrid peptide according to any one of the previous claims, characterized in that; it is used for the introduction of nucleic acids into cells.