TR202018990A2 - Methods for the production of gene products suitable for use in the treatment of hemophilia a disease - Google Patents
Methods for the production of gene products suitable for use in the treatment of hemophilia a disease Download PDFInfo
- Publication number
- TR202018990A2 TR202018990A2 TR2020/18990A TR202018990A TR202018990A2 TR 202018990 A2 TR202018990 A2 TR 202018990A2 TR 2020/18990 A TR2020/18990 A TR 2020/18990A TR 202018990 A TR202018990 A TR 202018990A TR 202018990 A2 TR202018990 A2 TR 202018990A2
- Authority
- TR
- Turkey
- Prior art keywords
- cells
- feature
- transgenic
- gene
- cd45rot
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000011282 treatment Methods 0.000 title claims abstract description 20
- 208000009292 Hemophilia A Diseases 0.000 title claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title abstract description 4
- 208000031220 Hemophilia Diseases 0.000 title description 2
- 201000010099 disease Diseases 0.000 title description 2
- 230000009261 transgenic effect Effects 0.000 claims abstract description 36
- 108010054218 Factor VIII Proteins 0.000 claims abstract description 25
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 claims abstract description 12
- 102100026735 Coagulation factor VIII Human genes 0.000 claims abstract description 11
- 201000003542 Factor VIII deficiency Diseases 0.000 claims abstract description 11
- 238000004113 cell culture Methods 0.000 claims abstract description 8
- 239000003550 marker Substances 0.000 claims abstract description 8
- 238000000338 in vitro Methods 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 76
- 102000001690 Factor VIII Human genes 0.000 claims description 18
- 229960000301 factor viii Drugs 0.000 claims description 17
- 210000001519 tissue Anatomy 0.000 claims description 11
- 238000002955 isolation Methods 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 108091008053 gene clusters Proteins 0.000 claims description 5
- 238000002513 implantation Methods 0.000 claims description 5
- 238000010255 intramuscular injection Methods 0.000 claims description 5
- 239000007927 intramuscular injection Substances 0.000 claims description 5
- 238000001990 intravenous administration Methods 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 238000007920 subcutaneous administration Methods 0.000 claims description 5
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 claims description 4
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 3
- 238000001802 infusion Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 238000002054 transplantation Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 2
- 108090000631 Trypsin Proteins 0.000 claims description 2
- 210000000577 adipose tissue Anatomy 0.000 claims description 2
- 239000011324 bead Substances 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 230000004663 cell proliferation Effects 0.000 claims description 2
- 210000005229 liver cell Anatomy 0.000 claims description 2
- 238000002690 local anesthesia Methods 0.000 claims description 2
- 238000010899 nucleation Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 239000012588 trypsin Substances 0.000 claims description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 2
- 238000005538 encapsulation Methods 0.000 claims 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 238000012217 deletion Methods 0.000 claims 1
- 230000037430 deletion Effects 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 claims 1
- 230000001954 sterilising effect Effects 0.000 claims 1
- 241000713666 Lentivirus Species 0.000 abstract description 4
- 241000701161 unidentified adenovirus Species 0.000 description 10
- 238000012546 transfer Methods 0.000 description 9
- 239000013598 vector Substances 0.000 description 8
- 230000028327 secretion Effects 0.000 description 7
- 238000001415 gene therapy Methods 0.000 description 6
- 230000000735 allogeneic effect Effects 0.000 description 5
- 239000002775 capsule Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 108700019146 Transgenes Proteins 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000015788 innate immune response Effects 0.000 description 3
- 238000007918 intramuscular administration Methods 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 241000700605 Viruses Species 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010071690 Prealbumin Proteins 0.000 description 1
- 239000008156 Ringer's lactate solution Substances 0.000 description 1
- 102000009190 Transthyretin Human genes 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Bu buluş; Faktör VIII gen dizilimini kodlayan bir promotör ve CD45ROt yüzey markörü taşıyan lentivirüslerin in vitro olarak elde edilmesi (i), transgenik hale dönüştürülecek hücrelerin dokudan izole edilmesi (ii), izole edilen hücrelerin bahsi geçen lentivirüslerle enfekte edilmesi (iii), enfekte edilen hücrelerin çoğaltılması (iv), hücre kültürü içerisinden CD45ROt eksprese eden hücrelerin ayrılarak izole edilmesi (v), izole edilen hücrelerin çoğaltılması (vi) adımlarını içeren hemofili A hastalığının tedavisinde kullanıma uygun gen ürünlerinin üretimi için bir yöntem ile ilgilidir.This invention; Obtaining lentiviruses carrying a promoter encoding the factor VIII gene sequence and a CD45ROt surface marker in vitro (i), isolating cells to be transformed into transgenic form from tissue (ii), infecting the isolated cells with the aforementioned lentiviruses (iii), multiplying the infected cells ( iv) it relates to a method for the production of gene products suitable for use in the treatment of hemophilia A disease comprising the steps of separating and isolating cells expressing CD45ROt from cell culture (v) growing the isolated cells (vi).
Description
TARIFNAME HEMOFILI A HASTALIGININ TEDAVISINDE KULLANIMA UYGUN GEN URUNLERININ URETIMINE ILISKIN Y`ONTEMLER Teknik Alan Bu bulus; hemofili A hastaliginin tedavisinde intramasküler, intravenöz vei'veya subkutan olarak uygulanabilen, otolog veya allojenik kullanima imkan veren gen ürünleri ve bunlarin üretim yöntemleri ile ilgilidir. DESCRIPTION GENE PRODUCTS SUITABLE FOR USE IN THE TREATMENT OF HEMOFILI A DISEASE METHODS OF PRODUCTION Technical Area This invention; Intramuscular, intravenous vei'or subcutaneous injection for the treatment of hemophilia A Gene products and their products that can be administered as an autologous or allogeneic related to production methods.
Teknigin Bilinen Durumu Klasik hemofili olarak da bilinen hemofili A, kanin pihtilasmasinda gereken Faktör VIII adli proteini üreten genlerdeki bozukluk olarak tanimlanmaktadir. Bu rahatsizligin görüldügü hastalarda Faktör VIII kanda çok azdir ya da hiç yoktur. State of the Art Hemophilia A, also known as classical hemophilia, is called Factor VIII, which is required for blood to clot. It is defined as a defect in the genes that produce the protein. Seeing this discomfort Patients have little or no Factor VIII in their blood.
Hemofili A tedavisinde gen tedavileri klinik test asamasinda çalisilan tedavilerdir. Mevcut teknikte uygulanan hemofili A gen tedavilerinin çogunda genler, adenovirüsler veya aktariminin çogunlukla intravenöz veya intramasküler olarak uygulanmasina olanak vermekte; tek bir yolla gen transferinin gerçeklestirilmesi de istenilen verimi saglamamaktadir. Gene therapies in the treatment of hemophilia A are treatments that are studied in the clinical testing phase. Available genes, adenoviruses or It allows the transfusion of the drug to be administered mostly intravenously or intramuscularly. giving; Realization of gene transfer in a single way also gives the desired efficiency. it does not provide.
A=nin tedavisinde kullanima uygun bilesimlerden ve bunlarin uygulanma rejimlerinden bahsedilmektedir. Söz konusu bilesimler; rekombinant adenovirüs ile birlikte Faktör Vlllinin ekspresyonunu saglayan bir promotör ve bir transtiretin güçlendirici içermektedir. Compounds suitable for use in the treatment of A = and their administration regimens is mentioned. The said compositions are; Factor with recombinant adenovirus It contains a promoter and a transthyretin enhancer that enables the expression of vllin.
Adenovirüslerin kullanimina örnek teskil eden bir diger doküman olan TR2019/11827 sayili patent dokümaninda bir yönde bir birinci transgene ve ters yönde bir ikinci transgene islevsel olarak bagli çift yönlü fare CMV (mCMV) promotörü içeren rekombinant adenovirüs (rAd) ve rAd vektörleri açiklanmaktadir. Bulus ayrica bu tür rAd ve rAd vektörlerini yapma ve kullanma yöntemlerinden bahsetmektedir. TR2019/11827, another document that exemplifies the use of adenoviruses a first transgene in one direction and a second transgene in the opposite direction. recombinant containing a bidirectional mouse CMV (mCMV) promoter operably linked to the transgene adenovirus (rAd) and rAd vectors are described. The invention also includes such rAd and rAd It talks about the methods of making and using vectors.
Ancak adenovirüs enjeksiyonunun gerçeklestirildigi tedavi yöntemlerinde dogal immünitenin gösterdigi direnç adenovirüsün etkinligini azaltmaktadir (Gregory SM. ve arkadaslari, Implications of the innate Immune response to adenovirus and adenoviral vectors, 2011; Hackett N.R ve arkadaslari, Adenovirus vectors for gene therapy, 2008). However, in treatment methods where adenovirus injection is performed, natural resistance of immunity reduces the effectiveness of adenovirus (Gregory SM. and friends, Implications of the innate Immune response to adenovirus and adenoviral vectors, 2011; Hackett N.R et al., Adenovirus vectors for gene therapy, 2008).
Virüslerle gen aktarimi dogal immünite olmasa dahi, süreç içerisinde gen aktarimi sirasinda kullanilan virüse karsi gelisecek olan immünite, aktarilan genin kaliciligini ve etkinligini olumsuz yönde etkilemektedir (Hackett NR ve arkadaslari, Antivector and antitransgene host responses in gene therapy, 2000; Bessis N. ve arkadaslari, Immune responses to gene therapy vectors: influence on vector function and effector mechanisms, 2004). Even if gene transfer with viruses is not natural immunity, gene transfer in the process The immunity that will develop against the virus used during negatively affects its effectiveness (Hackett NR et al., Antivector and antitransgene host responses in gene therapy, 2000; Bessis N. et al., Immune responses to gene therapy vectors: influence on vector function and effector mechanisms, 2004).
Adenovirüs enjeksiyon teknolojisinin bir diger dezavantaji da, aktarilan genin DNA'ya entegre olamamasindan kaynakli olarak, gen aktarimi etkinliginin aktarildigi hücre yasami ile sinirli kalmasidir. Buna göre gen aktarimi yapilan hücreler bölündükçe transgenik Faktör VIII ekspresyonu ve salinimi kaybolacaktir (Lee 08. ve arkadaslari, Adenovirus- Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine, 2017; Dani S.U., The challenge of vector development in gene therapy. Braz J Med Biol Res, 1999; Dunbar C.E. ve arkadaslari, Gene therapy comes of age, 2018). Another disadvantage of adenovirus injection technology is that the transferred gene cannot be transferred to DNA. cell life, where gene transfer efficiency is transferred, due to its inability to integrate to stay angry with. Accordingly, as transgenic cells divide, they become transgenic. Factor VIII expression and release will be lost (Lee 08. et al., Adenovirus- Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine, 2017; Dani S.U., The challenge of vector development in gene therapy. Braz J Med Biol Res, 1999; Dunbar C.E. and friends, Gene therapy comes of age, 2018).
Ayrica teknikte yer alan uygulamalarda Faktör VIII gen aktarimi yapildiktan sonra gen aktariminin etkinligi sadece genin fonksiyonu yani ekspresyon düzeyi ile takip edilebilmekte olup hücresel düzeyde bir marker ile izlenememektedir. Gen aktarim teknolojilerinde hücrelerin kullanildigi hayvan modelleri olmakla birlikte transgenik hücreler süspansiyon hücreler halinde verilmekte olup, süspanse hücrelerin dokuya implantasyonu ve çogalmasi mikroçevre nedeniyle istenilen sonucu verememektedir. Ayrica mevcut uygulamalar ile gerçeklestirilen gen aktariminda allojenik hücrelere karsi olusan immün yanit sebebiyle gen aktarimi kisa ömürlü olmaktadir. In addition, after the Factor VIII gene transfer is performed in the applications in the technique, the gene The efficiency of its transmission is only followed by the function of the gene, that is, its expression level. and cannot be monitored with a marker at the cellular level. gene transfer Although there are animal models in which cells are used in technologies, transgenic cells Suspension is given as cells and implantation of suspended cells into tissue and its proliferation cannot give the desired result due to the microenvironment. Also available Immune formation against allogeneic cells in gene transfer performed with applications gene transfer is short-lived due to the response.
Mevcuttaki uygulamalar incelendiginde, hemofili A hastaliginin tedavisinde kullanima uygun bilesimlerin ve yöntemlerin düsük etkinlige sahip oldugu ve yani sira birçok komplikasyon getirdigi görülmektedir. Bu dogrultuda; hemofili A'nin tedavisinde kullanima uygun olarak hem otolog hem de allojenik hücrelerin kullanimina olanak veren, dogal immün yaniti en aza indirgeyen ve Faktör Vlll sekresyonunu arttiran yenilikçi gen ürünlerine halen ihtiyaç oldugu düsünülmektedir. When the current applications are examined, it is possible to use it in the treatment of hemophilia A. suitable compositions and methods have low efficiency, as well as many appears to cause complications. In this direction; Use in the treatment of hemophilia A natural, which allows the use of both autologous and allogeneic cells in accordance with Innovative gene that minimizes immune response and increases Factor Vlll secretion products are still considered to be needed.
Bulusun Kisa Açiklamasi Mevcut bulus; yukarida bahsedilen gereksinimleri karsilayan, tüm dezavantajlari ortadan kaldiran ve ilave bazi avantajlar getiren, hemofili A hastaliginin tedavisinde kullanima uygun gen ürünlerinin üretimine iliskin yöntemler ile ilgilidir. Brief Description of the Invention The present invention; meeting the above-mentioned requirements, eliminating all the disadvantages It can be used in the treatment of hemophilia A disease, which removes and brings some additional advantages. relates to methods for the production of suitable gene products.
Bulusun öncelikli amaci; hemofili A hastaliginin tedavisinde kullanima uygun, hem otolog hem de allojenik hücrelerin kullanimina olanak veren, dogal immün yaniti en aza indirgeyerek Faktör VIII sekresyonunu arttiran gen ürünleri gelistirmektir. The primary purpose of the invention is; Suitable for use in the treatment of hemophilia A, both autologous as well as minimizing the innate immune response, which allows the use of allogeneic cells. to develop gene products that increase Factor VIII secretion by reducing
Bulusun bir diger amaci; mezenkimal kök hücreleri, endotel hücreleri ve karaciger hücrelerinin lentivirüslerle enfekte edilerek ex vivo sartlarda çogaltilmasiyla elde edilen gen ürünleri gelistirmektir. Another purpose of the invention; mesenchymal stem cells, endothelial cells and liver obtained by infecting cells with lentiviruses and multiplying them under ex vivo conditions. to develop gene products.
Bulusun bir diger amaci; gen konstraktinda yer alan inaktif CD45ROt (CD45RO truncated) yüzey markörü vasitasiyla faktör VIII sekresyonu takip edilebilen hücreleri içeren gen ürünleri gelistirmektir. Another purpose of the invention; inactive CD45ROt (CD45RO truncated) involved in the gene construct gene containing cells whose secretion of factor VIII can be tracked by a surface marker to develop products.
Bulusun bir diger amaci; süspanse, sferoid veya enkapsüle sferoid yapida elde edilebilen gen ürünlerinin gelistirilmesidir. Another purpose of the invention; Available in suspended, spheroid or encapsulated spheroid structure development of gene products.
Bulusun diger bir amaci, intravenöz ve/veya intramasküler vei'veya subkutan yollarla transplantasyona uygun gen ürünleri gelistirmektir. Another object of the invention is intravenous and/or intramuscular and subcutaneous routes. To develop gene products suitable for transplantation.
Bulusun diger bir amaci, hücre sagkalim orani ve fonksiyonelligi arttirilmis gen ürünleri gelistirmektir. Another object of the invention is gene products with enhanced cell survival rate and functionality. is to develop.
Bulusun bir diger amaci da; söz konusu gen ürünlerinin elde edilmesi için üretim yöntemlerinin gelistirilmesidir. Another purpose of the invention is; production to obtain said gene products development of methods.
Yukarida anlatilan amaçlarin yerine getirilmesi için bulus hemofili A hastaliginin tedavisinde kullanima uygun gen ürünlerinin üretimi için bir yöntem olup, i. Faktör VIII gen dizilimini kodlayan bir promotör ve CD45ROt yüzey markörü tasiyan Ientivirüslerin in vitro olarak elde edilmesi ii. Transgenik hale dönüstürülecek hücrelerin dokudan izole edilmesi iii. Izole edilen hücrelerin bahsi geçen lentivirüslerle enfekte edilmesi iv. Enfekte edilen hücrelerin çogaltilmasi v. Hücre kültürü içerisinden CD45ROt eksprese eden hücrelerin ayrilarak izole edilmesi vi. Izole edilen hücrelerin çogaltilmasi adimlarini içermektedir. To fulfill the above-described purposes, the invention is intended for the treatment of hemophilia A. It is a method for the production of gene products suitable for use in the treatment of I. A promoter encoding the factor VIII gene sequence and the CD45ROt surface marker In vitro recovery of carrier Ientiviruses ii. Isolation of cells to be transgenic from tissue iii. Infecting the isolated cells with the aforementioned lentiviruses iv. Multiplication of infected cells v. Isolation of cells expressing CD45ROt from cell culture to be made vi. Proliferation of isolated cells contains the steps.
Bulusun yapisal ve karakteristik özellikleri ve tüm avantajlari asagida verilen detayli açiklama sayesinde daha net olarak anlasilacaktir. Bu nedenle degerlendirmenin, detayli açiklama göz önüne alinarak yapilmasi gerekmektedir. The structural and characteristic features of the invention and all its advantages are detailed below. It will be clearer with the explanation. Therefore, detailed evaluation must be made taking into account the description.
Bulusun Detayli Açiklamasi Bu detayli açiklamada, bulus konusu hemofili A hastaliginin tedavisinde kullanima uygun gen ürünlerinin üretimine iliskin yöntemlerin tercih edilen yapilanmalari, konunun daha iyi anlasilmasina yönelik olarak ve hiçbir sinirlayici etki olusturmayacak sekilde açiklanmaktadir. Detailed Description of the Invention In this detailed description, the subject of the invention is suitable for use in the treatment of hemophilia A. Preferred embodiments of the methods for the production of gene products, for understanding and without any limiting effect is explained.
Bulus konusu, hemofili A hastaliginin tedavisinde kullanima uygun gen ürünlerinin üretimi için bir yöntem olup; i. Faktör VIII gen dizilimini kodlayan bir promotör ve CD45ROt yüzey markörü tasiyan Ientivirüslerin in vitro olarak elde edilmesi ii. Transgenik hale dönüstürülecek hücrelerin dokudan izole edilmesi iii. Izole edilen hücrelerin bahsi geçen Ientivirüslerle enfekte edilmesi iv. Enfekte edilen hücrelerin çogaltilmasi v. Hücre kültürü içerisinden CD45ROt eksprese eden hücrelerin ayrilarak izole edilmesi vi. Izole edilen hücrelerin çogaltilmasi adimlarini içermektedir. The subject of the invention is the production of gene products suitable for use in the treatment of hemophilia A. is a method for I. A promoter encoding the factor VIII gene sequence and the CD45ROt surface marker In vitro recovery of carrier Ientiviruses ii. Isolation of cells to be transgenic from tissue iii. Infecting the isolated cells with the aforementioned Ientiviruses iv. Multiplication of infected cells v. Isolation of cells expressing CD45ROt from cell culture to be made vi. It includes the steps of multiplying the isolated cells.
Bulus konusu yöntemde, (i) nolu islem adiminda bahsi geçen Ientivirüsler, Faktör VIII sekresyonunu saglayan bir promotör ve sekresyon yapan hücrelerin belirlenmesini saglayan CD45ROt yüzey markörü içeren gen kümeleri seklindedir. In the method of the invention, the entiviruses mentioned in the process step (i), Factor VIII Identification of a secretory promoter and secreting cells in the form of gene clusters containing the CD45ROt surface marker.
Bulusun tercih edilen uygulamasinda, bahsi geçen promotör Faktör VIII veya EF1 alpha seklindedir. Buna göre elde edilen gen kümeleri de ayni sirayla LV-Pf8-F8-CD45ROt veya LV-EF'ioi-FS-CD45ROt seklinde olmaktadir. Bu dogrultuda (i) nolu islem adiminda öncelikle plazmidlerin E. coli'ye transferi ile plazmid DNA üretimi ve bunu takiben in vitro olarak LV-Pf8-F8-CD45ROt veya LV-EF10i-F8-CD45ROt Ientivirüslerin üretimi gerçeklestirilmektedir. In the preferred embodiment of the invention, said promoter is Factor VIII or EF1 alpha. is in the form. Accordingly, the gene clusters obtained are LV-Pf8-F8-CD45ROt or It is in the form of LV-EF'ioi-FS-CD45ROt. In this direction, in the process step (i) Firstly, plasmid DNA production by transfer of plasmids to E. coli followed by in vitro production of LV-Pf8-F8-CD45ROt or LV-EF10i-F8-CD45ROt Ientiviruses is carried out.
Bulus konusu yöntemde, Faktör VIII gen diziliminin B kümesi (domain'i) çikartilmis haldeki tüm gen bölgesi lentiviral vektör (modifiye Ientivirüs) içerisinde yer almaktadir. Böylelikle fonksiyonel Faktör VIII proteininin daha etkin bir oranda eksprese edilmesi mümkün olmaktadir. In the method of the invention, the B cluster (domain) of the Factor VIII gene sequence is extracted. the entire gene region is contained in the lentiviral vector (modified Ientivirus). Thus, possible to express functional Factor VIII protein at a more efficient rate is happening.
Bulusun bir uygulamasinda göre (ii) nolu islem adiminda bahsi geçen doku; hastadan alinan yag dokusu, kemik iligi dokusu ve kan dokusunu içeren gruptan seçilmektedir. Bu uygulamaya göre söz konusu dokudan izole edilen hücreler ise; damar endotel hücreleri, karaciger hücreleri ve mezenkimal kök hücrelerden (MKH) en az biri olmaktadir. According to an embodiment of the invention, the tissue mentioned in the processing step (ii); from the patient The adipose tissue taken is selected from the group consisting of bone marrow tissue and blood tissue. This According to the application, the cells isolated from the said tissue are; vascular endothelial cells, liver cells and at least one of the mesenchymal stem cells (MSCs).
Bulusa iliskin (iii) nolu adimda ise; (ii) nolu adimda izole edilen hücreler (i) nolu adimda elde edilen Ientivirüsler ile inkübe edilerek enfekte edilmektedir. Bu sayede transgenik Faktör VIII salgilayan ve CD45ROt yüzeysel protein ekspresyonu gerçeklestiren transgenik hücreler elde edilmekte ve (iv) nolu adimla elde edilen bu hücrelerin bir hücre kültürü olusturularak sayica artmasi saglanmaktadir. (v) nolu islem adiminda ise gen kümesine eklenen inaktif CD45ROt markörünün hücre yüzeylerinde eksprese edilmeye baslamasiyla beraber Faktör VIII sekresyonu yapabilecek hücreler genetigi degistirilmemis hücreler arasindan ayirt edilmektedir. Ayirt edilen transgenik hücreler pozitif seçilim için manyetik boncuk teknolojisi (magnetic head-based cell separation) kullanilarak transgenik olmayan hücrelerden ayrilarak izole edilmektedir. (vi) nolu islem adimiyla da, (v) nolu adimda izole edilen transgenik hücreler, fizyolojik oranda Faktör Vlll salimi yapan bir gen ürününe dönüstürülmek üzere, hücre miktari 1 X106." kg ila 10x105/ kg araliginda olana kadar çogaltilmaktadir. Bulusun bir uygulamasina göre hücre çogaltimi 2 boyutlu hücre kültür sartlarinda gerçeklestirilmektedir. In step (iii) regarding the invention; Cells isolated in step (ii) were isolated in step (i) It is infected by incubation with the obtained Ientiviruses. In this way, transgenic secreting factor VIII and superficial expression of CD45ROt protein transgenic cells are obtained and these cells obtained by step (iv) It is ensured that the culture is created and increased in number. In the process step (v), the inactive CD45ROt marker added to the gene cluster is detected in the cell. be able to secrete Factor VIII together with its expression on their surface. cells are distinguished from non-genetically modified cells. distinguished magnetic bead technology (magnetic head-based) for positive selection of transgenic cells isolated from non-transgenic cells using cell separation. With the processing step (vi), the transgenic cells isolated in the step (v) were physiologically The amount of cells to be converted into a gene product that releases Factor VIII at a high rate is 1 X106." kg to 10x105/kg. According to this method, cell proliferation is carried out under 2-dimensional cell culture conditions.
Bulusun bir uygulamasina (A) göre; (vi) adimi ile elde edilen ve fizyolojik oranda Faktör Vlll sekresyonu saglayacak dozajdaki transgenik hücre kültürünün süspanse edilmesiyle bulus konusu gen ürünü süspansiyon seklinde elde edilmektedir. Bu uygulamaya (A) göre transgenik Faktör VIII salgilayan hücrelerin verimlilik oranini degerlendirmek üzere CD45ROt yüzeysel protein ifadesi akim sitometri ile kontrol edilebilmektedir. According to an embodiment of the invention (A); Factor obtained by step (vi) and in physiological ratio By suspending the transgenic cell culture at the dosage that will provide VIII secretion The gene product of the invention is obtained as a suspension. According to this application (A) To assess the fertility rate of cells expressing transgenic Factor VIII CD45ROt superficial protein expression can be checked by flow cytometry.
Bulusun bu uygulamasinda (A) bahsi geçen gen ürünü (süspanse transgenik hücreler), intravenöz infüzsyon ve/veya intramasküler enjeksiyon ile kullanima uygundur. Bahsi geçen gen ürünü hastaya tercihen intravenöz ve intramasküler yolla eszamanli olarak uygulanmaktadir. En tercih edilen haliyle ise, bahsi geçen gen ürününün bir dozajinin ürün otolog hücrelerin kullanimina uygun olup, eszamanli olarak intravenöz ve intramasküler olarak uygulanmasinin Faktör VIII sekresyonunu arttirdigi görülmüstür. In this embodiment of the invention (A), the gene product (suspended transgenic cells) It is suitable for use by intravenous infusion and/or intramuscular injection. bet The passed gene product is administered simultaneously to the patient, preferably intravenously and intramuscularly. is being implemented. Most preferably, a dosage of said gene product The product is suitable for the use of autologous cells and can be administered simultaneously intravenously and Intramuscular administration has been shown to increase Factor VIII secretion.
Bulusun bir diger uygulamasina (B) göre; (vi) adimi ile elde edilen ve fizyolojik oranda Faktör VIII sekresyonu saglayacak dozajdaki transgenik hücrelerden, 3 boyutlu kültür sartlarinda sferoid yapilar üretilmekte ve bulus konusu gen ürünü sferoid transgenik hücreler seklinde elde edilmektedir. Bulusun bu uygulamasina (B) göre; yöntem a. %70-80 yogunluga erisen transgenik hücrelerin tripsin uygulamasiyla tabaktan kaldirilip fosfatIi-tuz tampon (PBS) çözeltisiyle yikanarak sayilmasi yapisma gerektirmeyen 96 kuyucuklu hücre kültür plakalarina ekim yapilmasi Ekilen hücrelerin 24 saat inkübe edilmesi d. Sferoid seklini alan hücrelerin ringer laktat solüsyonu ile yikanarak transplantasyona hazir hale getirilmesi alt islem adimlarini içermektedir. According to another embodiment of the invention (B); obtained by step (vi) and at a physiological rate 3D culture from transgenic cells at the dosage that will provide factor VIII secretion. conditions, spheroid structures are produced and the gene product of the invention is spheroid transgenic. obtained in the form of cells. According to this embodiment of the invention (B); method a. Transgenic cells reaching 70-80% density were removed from the plate by trypsin application. removed and counted by washing with phosphate-salt buffer (PBS) solution seeding into adhesion-free 96-well cell culture plates Incubation of seeded cells for 24 hours D. Washing the spheroid-shaped cells with Ringer's lactate solution getting ready for transplantation contains subprocess steps.
Bulusun bu uygulamasinda (B) bahsi geçen gen ürünü (sferoid transgenik hücreler), intramasküler enjeksiyon veya subkutan implantasyon ile kullanima uygundur. Bulusun bu uygulamasi (B) ile vücut içerisinde Faktör VIII üretiminin uzun süreli olarak korunmasi amaçlanmaktadir. Transgenik otolog hücrelerin vücuda sferoid yapida verilmesini mümkün kilan bulus konusu gen ürünü sayesinde fonksiyonellik ve kalicilik önemli oranda arttirilmaktadir. In this embodiment of the invention (B), the gene product mentioned (spheroid transgenic cells), It is suitable for use by intramuscular injection or subcutaneous implantation. find this long-term preservation of Factor VIII production in the body is intended. Giving transgenic autologous cells into the body in a spheroid structure Thanks to the gene product of the invention, which makes it possible, functionality and permanence have increased significantly. is being increased.
Bulusun bir diger uygulamasina (C) göre; (B) uygulamasinda 3 boyutlu kültür sartlarinda sferoid yapida elde edilen transgenik hücreler üç boyutlu yazici ile üretilmis makrokapsüller içerisine enkapsüle edilmekte ve bulus konusu gen ürünü enkapsüle sferoid transgenik hücreler seklinde elde edilmektedir. Bu uygulamaya (C) göre sferoid halini alan hücreler sterilize edilerek kapsül içerisine enjekte edilmektedir. Tercihen bir de 10-50, tercihen ortalama 30 sferoid bulunmaktadir. According to another embodiment of the invention (C); (B) in 3D culture conditions Transgenic cells obtained in spheroid structure were produced with a three-dimensional printer. are encapsulated into macrocapsules and the gene product of the invention is encapsulated. It is obtained as spheroid transgenic cells. Spheroid according to this application (C) The cells that have formed are sterilized and injected into the capsule. Preferably a There are 10-50 spheroids, preferably an average of 30.
Bulusun bu uygulamasiyla (C) elde edilen gen ürünü (enkapsüle sferoid transgenik hücreler) lokal anestezi altinda subkutan implantasyon ile kullanima uygun olup ayrica otolog hücrelerin yani sira allojenik hücrelerin naklini de mümkün kilmaktadir. Bulus konusu bu ürün, hastanin ihtiyaç duydugu doz dikkate alinarak kapsüller halinde deri altina yerlestirilmektedir. Bulus konusu yöntemin bu uygulamasi ile elde edilen gen ürünü kapsül formunda oldugundan, hücrelerin canliliklarini ve fonksiyonelliklerini sürdürmeleri için biyomimetik bir üçboyutlu ortam saglamaktadir. Bu sayede nakledilen hücrelerin immün yanitina bagli olarak parçalanmasina engel olmakta ve nakledilen hücrelerin hasta vücudundaki fonksiyonlari iyilestirilmekte ve yasam süreleri uzatilmaktadir. Bununla birlikte gen ürününün kapsül formunda olmasi her an satisa hazir halde depolanmasina da olanak saglamaktadir. The gene product (C) obtained by this embodiment of the invention (encapsulated spheroid transgenic cells) are suitable for use with subcutaneous implantation under local anesthesia and also It makes it possible to transplant allogeneic cells as well as autologous cells. Meet This product in question is administered to the skin in capsules, taking into account the dose needed by the patient. is placed underneath. Gene product obtained by this application of the method of the invention Since it is in capsule form, cells can maintain their vitality and functionality. It provides a biomimetic three-dimensional environment for In this way, the transplanted cells It prevents the disintegration of the transplanted cells due to the immune response and functions in the body are improved and life spans are extended. With this However, the fact that the gene product is in capsule form allows it to be stored ready for sale at any time. also provides opportunities.
Bulus kapsamindaki tüm uygulamalarda kullanilan Ientivirüslere karsi dogal immünite gelisimi mevcut teknikte kullanilan adenovirüslerle kiyaslandiginda çok daha nadir görülmektedir. Bunun yani sira bulus konusu gen ürünü kapsaminda gelistirilen uygulamalarla da immünitenin tedavi sürecindeki etkileri minimize edilmekte, Faktör VIII sekresyonu ve hücrelerin sagkalim oranlari arttirilmaktadir. Innate immunity against Ientiviruses used in all applications within the scope of the invention development is much rarer when compared to adenoviruses used in the current art. is seen. In addition, the gene product developed within the scope of the invention The effects of immunity in the treatment process are minimized with these applications, Factor VIII secretion and survival rates of cells are increased.
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR2020/18990A TR202018990A2 (en) | 2020-11-25 | 2020-11-25 | Methods for the production of gene products suitable for use in the treatment of hemophilia a disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR2020/18990A TR202018990A2 (en) | 2020-11-25 | 2020-11-25 | Methods for the production of gene products suitable for use in the treatment of hemophilia a disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| TR202018990A2 true TR202018990A2 (en) | 2021-02-22 |
Family
ID=75577024
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TR2020/18990A TR202018990A2 (en) | 2020-11-25 | 2020-11-25 | Methods for the production of gene products suitable for use in the treatment of hemophilia a disease |
Country Status (1)
| Country | Link |
|---|---|
| TR (1) | TR202018990A2 (en) |
-
2020
- 2020-11-25 TR TR2020/18990A patent/TR202018990A2/en unknown
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104204194B (en) | Produce the method for natural killer cells, by the natural killer cells of this method production and the composition for treating cancer and infectious diseases comprising the natural killer cells | |
| Itoh et al. | Age-related variation in the proportion and activity of murine liver natural killer cells and their cytotoxicity against regenerating hepatocytes. | |
| DE69633668T2 (en) | ALLOGENIC CELL THERAPY FOR CANCER DUE TO ALLOGENIC STEM CELLS TRANSPLANTATION | |
| CN103212071B (en) | Stem cell fusion model of carcinogenesis | |
| El-Tahawy et al. | Effect of platelet rich plasma (PRP) injection on the endocrine pancreas of the experimentally induced diabetes in male albino rats: a histological and immunohistochemical study | |
| US20080057043A1 (en) | Monocyte cell | |
| BR112016009753B1 (en) | IN VITRO METHODS FOR CULTURE OF MESENCHYMAL STROMA CELL SAMPLES AND FOR THE PREPARATION OF MESENCHYMAL STROMAL CELLS | |
| CN108697777A (en) | To the pretreatment drug of the T cell infusion therapy of immunologic test point inhibitor repellence tumour | |
| KR20090033328A (en) | Immune privileged and modulatory progenitor cells | |
| CN105769910A (en) | Application of human amniotic mesenchymal stem cells | |
| US20230210897A1 (en) | Modified natural killer cells and methods of using the same | |
| CN104349785A (en) | Isolation and use of human lymphoid organ-derived inhibitory stromal cells | |
| ES2377464T3 (en) | Cell therapy: a method and composition to treat diabetes | |
| CN104736709A (en) | Compositions and treatments based on cadherin modulation | |
| CN106867963A (en) | Ray modification umbilical cord adult stem cell 3D microballoon work preparation and its preparation and application | |
| CN104232570B (en) | Set up the method and its application of monoclonal mescenchymal stem cell | |
| CN105963699B (en) | Target spot and application of the FATS as melanoma immunization therapy | |
| CN109562145A (en) | The pharmaceutical composition and its application of insulin-containing like growth factor -2 | |
| CN110035742A (en) | Stem cell biomimetic particles | |
| CN109789187A (en) | Purposes of the IL-12 as substitution immunotherapeutic agent | |
| TR202018990A2 (en) | Methods for the production of gene products suitable for use in the treatment of hemophilia a disease | |
| CN103911342A (en) | Mice animal model of giant cell tumor of bone of human and construction method thereof | |
| CN112999211B (en) | Application of ceramide molecule in preparation of medicine for inhibiting esophageal squamous cell carcinoma metastasis | |
| CN104434973A (en) | Method for intensifying functions of cytokine-induced killer cells | |
| CN103784940A (en) | Method and reagent for improving hematopoietic stem cell homing and implanting rate |