CN111100847A

CN111100847A - Telomere gene therapy product with higher safety for human health to reverse aging

Info

Publication number: CN111100847A
Application number: CN201911262488.4A
Authority: CN
Inventors: 刘玉文
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2020-05-05

Abstract

In human cells, cells divide once per minute, telomeres at the top of chromosomes are shortened once, and when the telomeres can not be shortened any more, the cells can not divide continuously. At this time the cells reached the generally accepted limit of 100 divisions and began to die. Therefore, the telomere is regarded as a 'life clock' telomere by scientists and has the important functions of protecting chromosome stability and identifying a starting point of cell renewal and division, the length of the telomere reaches the functions of a promoter and a silencer and is used for regulating and controlling the expression of proteins at the downstream of a chromosome, the expression and aging of the proteins are related, and the lengthening of the telomere is very important for health reversal aging. The adenovirus gene is used as a treatment vector, and the activity of telomerase is strictly controlled, so that the carcinogenic risk is reduced to zero. In order to solve the production problem of telomere gene therapy vector, a method for packaging adenovirus gene therapy vector in batches is added.

Description

Telomere gene therapy product with higher safety for human health to reverse aging

The invention belongs to the field of the following:

the present invention is based on the results of the inventions in the technical field of gene therapy and the technical field of gene engineering.

Technical background:

gene therapy is the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes, in order to achieve therapeutic goals. Also includes the technical application of transgenosis and the like. That is, the foreign gene is inserted into an appropriate recipient cell of a patient by gene transfer technology so that the product produced by the foreign gene can treat a disease. In a broad sense, gene therapy may also include measures and new technologies taken from the DNA level to treat certain diseases.

Somatic gene therapy: somatic gene therapy is the transfer of normal genes into somatic cells to express gene products for therapeutic purposes. The ideal measure of the method is to introduce exogenous normal genes into specific gene loci of chromosomes in target somatic cells, and exactly replace abnormal genes with healthy genes so as to ensure that the abnormal genes play a therapeutic role.

The virus mediated gene transfer is realized by a conversion mode, namely, the virus is taken as a vector, an exogenous target gene is assembled on the virus by a gene recombination technology, and the recombinant virus is used for infecting host cells of a receptor, and is called as a virus carrier.

DNA virus-mediated vector: it has been found that adenoviruses which are replication-defective due to the absence of the E1 region can be propagated in cells expressing the E1 gene. Later, it was demonstrated that replication-defective adenoviruses carrying exogenous DNA exhibit the same reproductive characteristics. In 1993, America and other countries successfully adopt adenovirus vectors to carry out in vivo gene transfer of heart, brain, lung, intrahepatic bile ducts and muscle tissues. It represents a new direction for gene therapy, and the use of replication-defective adenoviruses for gene therapy has the following advantages:

① the virus can infect dividing and non-dividing cells, and can obtain a large amount of gene products, and has special significance for correcting gene defects such as nerve cells and myocardial cells;

② the virus particles are relatively stable and easy to purify and concentrate without reducing infectivity;

③ can be used to transduce target cells efficiently but less freely outside the cell genome, and can be expressed continuously;

④ Ad5 adenovirus subgroup C that has been used for gene therapy, is not carcinogenic.

The new adenovirus vector has the advantages that 48000bp gene can be successfully carried, while other viruses only can transport 7000bp gene. These advantages show the broad application prospects of adenovirus-mediated vectors.

The gene therapy targets:

telomeres are small fragments of DNA-protein complexes present at the end of the linear chromosomes of eukaryotic cells, which together with telomere binding proteins form a special "cap" structure that serves to maintain chromosome integrity and control the cell division cycle. Telomeres, centromeres and origins of replication are the three major elements of the chromosome that remain intact and stable.

Telomeres, the length of which reflects the history and replication potential of the cell, are called the "mitotic clock" of the cell's lifespan.

Telomeres are short, multiple, non-transcribed sequences (TTAGGG) and some binding proteins constitute a special structure, which, in addition to providing a buffer for non-transcribed DNA, can protect the ends of chromosomes from fusion and degeneration, play an important role in chromosome localization, replication, protection and control of cell growth and life, and are closely related to apoptosis, cell transformation and immortalization. When the cell divides once, the telomeres of each chromosome become shorter successively.

About 50-200 nucleotides of a part of genes constituting telomeres cannot be completely replicated (lost) due to multiple cell divisions, so that the cells stop their functions unstably.

The more cell division, the more telomere wear and the shorter the cell life.

Telomeric DNA is composed of simple highly repetitive sequences of DNA, with GT enriched at the ends of the chromosome along the strand in the 5 'to 3' direction. In yeast and human body, the telomere sequence is C1-3A/TG1-3 and TTAGGG/CCCTAA, respectively, and a plurality of proteins are combined with telomere DNA,

mode of action of telomerase

Telomeric DNA has major functions:

first, protecting chromosomes from nuclease degradation;

second, preventing chromosomes from fusing to each other;

and thirdly, providing a substrate for telomerase, solving the problem of tail end retraction of DNA replication and ensuring complete replication of chromosomes.

Telomeres, centromeres and origins of replication are the three major elements of the chromosome that remain intact and stable. Meanwhile, telomeres are special sites for gene regulation, and can often inhibit the transcriptional activity of genes located near the telomeres (called the position effect of the telomeres, TPE).

In most eukaryotes, elongation of telomeres is catalyzed by telomerase. In addition, recombination mechanisms also mediate lengthening of telomeres. Telomerase is an enzyme which is responsible for lengthening telomeres in cells, is a basic nucleoprotein reverse transcriptase, can add telomere DNA to the end of chromosome of a eukaryotic cell, fills up the telomeres lost by DNA replication, prolongs telomere repair, can prevent the loss of the telomeres due to cell division, and increases the number of times of cell division. Telomeres have important functions of maintaining chromosome stability and cell activity in cells of different species, and telomerase can prolong and shorten the telomeres (the shortened telomeres have limited cell replication capacity), so that the proliferation capacity of cells in vitro is enhanced. Telomerase activity is inhibited in normal human tissues and reactivated in tumors, potentially participating in malignant transformation. Telomerase has important effects in keeping telomere stable, complete genome, long-term activity of cells, potential continued proliferation capacity and the like. The existence of telomerase fills up the defect of DNA replication, namely, by repairing and prolonging telomeres, the telomeres can not be lost due to cell division, so that the number of cell division is increased. However, in normal human cells, telomerase activity is very tightly regulated, and active telomerase is detected only in hematopoietic, stem and germ cells, which must be constantly dividing. After the cells are differentiated and mature, the cells have to be responsible for the requirements of various tissues in the body, and each function is played, so that the activity of telomerase is gradually lost, the telomerase is an enzyme consisting of catalytic protein and an RNA template, DNA at the tail end of a chromosome can be synthesized, the cells are endowed with the immortality of replication, the old cells are, and the length of the telomere is shorter; the younger the cells are, the longer the telomeres are, and the telomeres are associated with cell aging. Some telomeres in senescent cells lose most of the telomeric repeats. Senescence occurs when the function of cell telomeres is impaired. When telomeres shorten to a critical length, senescence accelerates and death approaches.

The invention content is as follows:

the invention is a new treatment technology based on the patent of the inventor for health care and anti-aging therapy, the used gene therapy vector is an adenovirus vector, and the adenovirus is a macromolecular 36kb double-stranded non-enveloped DNA virus. It enters into cell through receptor-mediated endocytosis, then carries telomerase reverse transcriptase gene to transfer into nucleus to carry out gene therapy, keeps outside chromosome, does not integrate into host cell genome, vector only retains the necessary element Ad5 psi for adenovirus packaging, removes 5LTR and 3LTR elements with human body replication origin function, has no replication origin and is difficult to package into virus particle, but has coping scheme and batch production patent, uses replication origin completely different from human body cell, the replication origin can only play replication function in escherichia coli, completely loses replication function in human body cell, completes packaging in BJ5183 escherichia coli, is equipped with environment packaged by adenovirus, packaging adenovirus vector in bacteria has the advantages of no other wild virus, affects safety of therapeutic use vector, introduces gene replication factor required by escherichia coli in mammalian cell, the DNA polymerase and the helicase can be packaged, compared with the prior invention patents, the invention has the advantages of treatment, the invention controls the activity of telomerase gene more strictly, adds a guarantee, ensures that the risk of carcinogenesis is zero under multiple guarantees, the treatment effect is possibly discounted than the prior patent, because the CMV promoter has higher efficiency than TRE3G, the treatment efficiency has to be reduced for enough safety of treatment products, the treatment efficiency is reduced, the safety is improved, the telomerase gene is controlled in the activity of biological cells, part of the telomerase gene needs to be greatly updated to have activity for a long time, the probability of active carcinogenesis of the telomerase gene for a long time is still very low, the first guarantee of the invention is the same as the prior patent, the treatment vector has no replication origin of human cells, the replication origin of human cells needs specific conserved gene sequences, therefore, the treatment vector can not be replicated in the human cells, the treatment carrier can not passage cells, the divided cells do not carry treatment carrier genes, the treatment carrier genes are dissociated from cell chromosomes, the treatment carrier genes can be degraded and disappear in a certain time without the protection of nucleosomes, and the activity of telomerase genes is strictly controlled, so the prior patent technology is safe enough.

The second safety guarantee is added, the inducible promoter TRE3G which is regulated most strictly is used for expressing the telomerase gene, the Tet3G repressor protein is improved from rtTA repressor protein and has higher sensitivity, and the Tet3G regulation expression system changes the conformation of the regulatory protein through inducing drugs (such as tetracycline, doxycycline and the like) so as to achieve the purpose of regulating the expression of the target protein. The Tet3G regulated gene expression system is established based on the Tet resistance operon on the Tn10 transposon of Escherichia coli. The Tet3G repressor protein binds specifically to the TRE3G operon. When no induction drug exists, Tet3G binds to TRE3G, thereby blocking the expression of telomerase genes; when an induction drug exists, the conformation of Tet3G is changed and is separated from TRE3G, so that telomerase genes are expressed, the activity of telomerase is strictly controlled, and tetracycline drugs are stopped to be taken after the gene therapy vector finishes the lengthening treatment of cell telomeres, so that the activity of telomerase is not ensured, which is the second important guarantee.

Drawings

FIG. 1 adenovirus vector sequence characteristics:

FIG. 1 shows the functions of the elements in the vector, Ad5 Ψ is a recognition site of the vector gene packaging signal for packaging the vector into viral particles, the vector TRE3G inducible promoter and telomerase reverse transcriptase gene are used for drug-regulated transcription and translation of telomerase, telomerase enters the nucleus to complete the telomere elongation treatment of cells, the vector Tet3G is a function of turning on and off TREG by combining repressor protein of TRE3G promoter, the vector SV40poly (A) element is responsible for mRAM stability and access to the nuclear pore, the vector promoter element and AmpR element are responsible for screening Escherichia coli for packaging adenovirus particles, AmpR is only expressed in Escherichia coli, and has no effect on human cells when the expression function is not available in human cells, the vector ori element is an exclusive replication origin of Escherichia coli and is responsible for vector copying and packaging into adenovirus particles, and the ori replication origin of replication function in mammalian cells and other bacteria, or mammalian cells must be treated to provide an ori origin of replication environment for replication.

The invention contents of various gene therapy vectors are packaged in batches:

the Tet-On3G expression regulation system can also be used for packaging various viruses in bacteria and mammalian cells and is characterized in that pcr amplification genes are not used, packaging plasmids do not need to be introduced every time, all plasmids required for packaging are introduced into receptor cells, the expression of viral genes is controlled by the Tet-On3G expression regulation system, viral particles cannot be generated under the condition without tetracycline, the growth and the division of the receptor cells cannot be influenced, the amplified receptor cells are cultured, part of the receptor cells are taken out and added with tetracycline for culture, and then the viral genes are activated and packaged to generate the viral particles, so that various viral vectors can be packaged in batches. Both laboratories and biological companies package by pcr amplification and gene introduction, which is not only costly but also inefficient, experimental methods such as packaging a set of adenovirus packaging plasmids pAAV-RC2, pAAV-MCS, phepper together into adeno-associated virus, the vector of pAAV-RC2 contains CAP and REP genes, these genes can generate capsid and DNA replication proteins required for infectious virus production, co-transfect the vector into a packaging cell line to prepare recombinant replication-defective AAV virus, the pAAV-RC plasmid contains AAV-2REP and CAP genes, respectively encoding replication proteins and viral capsid structural proteins. Establishing the correct expression levels of the REP and CAP gene products is an important step in achieving high titer virus production. pAAV-RC guides the expression of REP and CAP from two different promoters, the two promoters are enzymatically cut off and switched into a TRE3G promoter to regulate the expression of REP and CAP, one or two of pAAV-MCS and pHelper are inserted with a Tet3G repressor gene, the two plasmids and a target gene vector are firstly introduced into a receptor cell, the receptor cell expresses enough Tet3G repressor after a period of time, pAAV-RC2 is introduced into the receptor cell, the receptor cell is cultured and amplified, a part of the receptor cell is taken out and added with tetracycline for culturing, the expression of REP and CAP is activated, and an adenovirus vector is packaged in the receptor cell.

The implementation mode is as follows:

the gene therapy product for lengthening telomeres needs a large amount of virus particles, the ideal effect can be achieved by enough quantity, the gene therapy product which is safe and non-toxic and has no influence on human bodies is purified, the quantity of the therapy vectors used for one-time therapy is about 5 trillion, which is about 10 percent of the quantity of human cells, the existing packaging technology is low in efficiency and can not be achieved or is high in cost, and in order to enable everyone to enjoy the benefit of therapy, the invention combines two vectors of the inventor to construct a package, and the therapy vector can be efficiently packaged. The immune function of a therapist is reduced, the therapist can have fever symptoms after inputting into a human body in a sterile medical room by a venous transfusion mode, the symptoms can be eliminated and the treatment and the immunity recovery can be completed after two days, the therapist also needs to take a tetracycline medicament for 1 month, a treatment carrier just enters human body cells, the treatment carrier can still achieve the treatment effect within one week without the tetracycline medicament because the treatment carrier can lose efficacy without the tetracycline medicament after one week, one-time gene treatment can complete the lengthening of the telomeres of hundreds of millions of cells, more than trillions of renewable dividing cells can be generated, the service life is prolonged by about one percent, the therapist only needs to perform one-time treatment in half a year, the health telomere can be ensured not to be worn, the health treatment effect of health reversion aging can be achieved, a series of aging diseases such as cancer and health risks brought by stroke can be generated along with the increase of the age of the body telomere, the predicted life-span improvement of long-term maintenance treatment can reach more than 50%, only telomeres need to be prolonged for a single cell strain in a laboratory, the cell is endowed with immortalization, the life-span improvement for human cells is the basis of cell updating, the most ideal anti-aging treatment is realized, the cell is also limited by 2% of highly differentiated non-dividing cells of a body, the stem cell regeneration treatment for the non-dividing cells is needed to break through the limit of the life span, certain achievements are achieved in the stem cell regeneration treatment world at present, and the invention has important application in the safety amplification culture of the stem cells.

Claims

1. One gene therapy use is an adenovirus particle comprising the telomerase reverse transcriptase gene.

2. Adenovirus therapeutic vectors do not contain a mammalian cell origin of replication.

3. The promoter of telomerase reverse transcriptase is TRE 3G.

The Tet3G repressor gene and the TRE3G inducible promoter constitute the regulation of telomerase reverse transcriptase activity.

5. The gene therapy products for prolonging and maintaining telomere gene of human cells as in 1, 2, 3 and 4 are characterized in that the products do not replicate after entering human cells, carrier genes do not passage cells, tetracycline regulates the activity of telomerase reverse transcriptase, avoids carcinogenesis, and expresses the telomerase reverse transcriptase to prolong the telomere gene, thereby achieving the treatment effects of health care and prolonging life.

6. The adenovirus vector comprises: ad5 Ψ gene, TRE3G gene, telomerase reverse transcriptase gene, CMV enhancer gene, CMV promoter gene, Sv40poly (A) gene, ori gene, AmpR promoter gene, AmpR gene.