Patent Publication Number: US-2022228166-A1

Title: Application of Lentiviral Vector EF1alpha Promoter for Optimising ABCD1 Gene Expression to Treat Adrenoleukodystrophy

Description:
TECHNICAL FIELD 
     The present application belongs to the field of gene engineering and, in particular, to an application of a lentiviral vector EF1α promoter for optimizing ABCD1 gene expression to treat adrenoleukodystrophy. 
     BACKGROUND 
     Adrenoleukodystrophy (ALD) is a recessive inherited lipid metabolic disease. This disease is caused by the massive accumulation of very long chain fatty acid (VLCFA) in organs and tissues such as blood, brain white matter, adrenal cortex due to dysfunction of peroxisomes in oxidating VLCFA in cells, resulting in central nervous system demyelination and adrenocortical atrophy or dysplasia. ALD is the most common peroxisomal disease, and mainly affects adrenal glands and cerebral white matter. More than half of patients start to suffer from this disease in their childhood or adolescence. The disease is mainly characterized by progressive psychomotor disorders, visual loss and hearing loss, and/or adrenal hypofunction. The incidence of this disease is about 0.5/100,000 to 1/100,000, 95% in male and 5% for female heterozygote. There is no region specificity and race specificity. 
     Since ALD is a disease caused by monogenic mutations, gene therapy can theoretically achieve complete treatment of this disease. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) have several characteristics that make them potential “vehicles” for gene therapy. HSCs and MSCs can be obtained from blood or bone marrow, and have the ability to differentiate into a series of somatic cells and update various tissue cells. However, if the ABCD1 gene expression is low in cells, the abnormal accumulation of VLCFA in cytoplasm could not be effectively removed. 
     Although there are various gene therapy methods for gene transfer by using viral vectors at home and abroad, the gene transfer efficiency is greatly different due to different viral vectors or even different preparation methods for the same vector, and the gene transfer efficiency directly affects the disease therapeutic effect. Most of the existing methods for treating genetic diseases by using cell therapy have much low efficiency and only modify blood stem cells, so that the clinical effect of disease treatment is not as expected. Therefore, a method for improving the viral gene transfer efficiency to the maximum extent and modifying various stem cells to improve the therapeutic effect of genetic diseases is urgently needed. 
     Conducted by BlueBird Bio (U.S.) and other institutes, clinical trials of gene therapy for 17 children suffering from ALD by using lentiviral vectors carrying normal ABCD1 genes are now in the data collection stage. Currently, the results show that a certain response effect to the therapy within two years occurs in patients, but the overall effect is still weak. Among patients, one patient loses his ability to act and speak, and the other cases are still under observation, so gene therapy for ALD disease is currently in the initial stage. 
     SUMMARY 
     The method provided by the solution of the present application is mainly to realize ALD gene therapy by transfecting autologous hematopoietic stem cells (HSCs) of a patient with an improved and optimized lentiviral vector carrying an ABCD1 gene under an EF1α promoter, where it may be carried out in combination with direct intracerebral injection of lentiviral vector carrying the ABCD1 gene according to actual conditions. The specific components of the carrier protein, the optimization and improvement of the packaging cells and purification mode are all directionally designed. Any other cases using same modified systems shall be considered as infringement, including changes of the promoter (such as replacing EF1α with CMV to realize gene expression), shortening of the length of ABCD1 proteins (such as using only partial sequences of functional domains) and addition of any meaningless protein to the lentiviral vector on the basis of this system. 
     One aspect of the present application discloses a lentiviral vector containing a recombinant plasmid and a preparation method thereof. The present application mainly optimizes the lentiviral vector itself and packaging and purification methods for ABCD1 gene expression. And, various stem cells are used as delivery vectors, so that the gene has high expression efficiency in differentiated or undifferentiated stem cells. The expression level of the ABCD1 gene in transgenic cells is significantly increased after optimization of the lentiviral vector. In addition, the lentiviral vector adopted in the present application is an updated gene transfer system, which allows, under the initiation of the EF1α promoter, more efficient gene transfer while ensuring safety. The lentiviral vector adopted in the present application is an optimized and improved gene transfer system, and compared with other previous systems, this system has higher transfection efficiency, stronger stability, and better safety, and can complete the transfer of normal genes more efficiently in the hemophilia gene therapy process. With transduction through this system, stable expression of the ABCD1 gene in HSCs and MSCs can be successfully realized, fully proving the feasibility of the present application on successful hemophilia gene therapy. 
     The present application provides a lentiviral vector carrying a normal ABCD1 gene, including an ABCD1 gene sequence, a human EF1a promoter sequence and an NHP/TYF lentiviral vector system. 
     The NHP/TYF lentiviral vector system includes vectors pNHP, pTYF and pHEF-VSV-G. 
     Another aspect of the present application discloses a stem cell system modified with the lentiviral vector. The sequence of the ABCD1 gene is shown in SEQ ID NO.1. The recombinant plasmid is an EF1a promoter sequence shown in SEQ ID NO.3 followed by the ABCD1 gene sequence shown in SEQ ID No.1. The lentiviral vector is obtained by connecting the recombinant plasmid to a lentiviral vector, followed by packaging, purifying and concentrating. The lentiviral vector-modified dual stem cell system is to transfect one or two kinds of peripheral hematopoietic stem cells or mesenchymal stem cells respectively with the lentiviral vector, and preferably, simultaneously transfect hematopoietic stem cells and mesenchymal stem cells to obtain a dual stem cell system which can stably express the ABCD1 gene. 
     The present application has the following advantages: The dual stem cells are modified with the lentiviral vector such that the stem cells can stably and massively express the ABCD1 gene, laying an important foundation for better improving the therapeutic effect of ALD treatment. In addition, with respect to treatment manners, according to the actual conditions of the patient, the present solution adopts a treatment means combined with a mode of transfecting the dual stem cells with the lentiviral vector carrying the ABCD1 gene and injecting the dual stem cells back intravenously, which is of important significance to realize the quicker ALD symptom relief and the more comprehensive and durable gene treatment. Therefore, in the present application, the ALD gene therapy is subjected to vector optimization and transgenic efficiency improvement, and compared with methods only using a stem cell line for treatment, this method uses the dual stem cell lines for treatment and thus can effectively improve the transfer efficiency and the expression level activity of the ABCD1 gene, and is of important significance to guarantee the effectiveness of gene therapy. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a schematic diagram illustrating the construction of a lentiviral vector and a production and purification process thereof; 
         FIG. 2  illustrates protein identification of the protein expression level of the ABCD1 gene in stem cells; 
         FIG. 3  is a schematic diagram illustrating a therapeutic procedure for treating ALD disease by using a dual stem cell system obtained through transfection with a lentiviral vector carrying a normal ABCD1; and 
         FIG. 4  is clinical case records of ALD treatment using dual stem cells (hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs)) obtained through transfection with the lentiviral vector carrying the normal ABCD1. 
     
    
    
     DETAILED DESCRIPTION 
     To further elaborate on the technical means adopted and the effects achieved in the present application, the technical solutions of the present application are specifically described below through specific examples in conjunction with drawings, but the present application is not limited to the scope of the examples. 
     The contents without specific techniques or conditions specified in the examples are conducted according to technical conditions described in the literature widely recognized in the art or according to specifications of the corresponding products. 
     The reagents or instruments used herein without manufacturers specified are conventional products commercially available from proper channels. 
     Example 1: Construction, Packaging and Purification of Lentiviral Vector Carrying Normal ABCD1 Gene 
     A normal ABCD1 gene sequence (shown in SEQ ID NO. 1) and a human EF1α promoter sequence (shown in SEQ ID NO. 3) were synthesized through gene synthesis, and connected into a lentiviral vector (NHP/TYF lentiviral vector system) through restriction enzyme sites. The obtained product was identified through manners such as sequencing and double enzyme digestion (a BamHI cloning site ggatccacc-AUG was used for 5′, and a SpeI site was used for 3′ for cloning; and optimal reaction conditions were referred to the original NEB suggestion) to obtain a correctly connected lentiviral expression vector which carried a normal ABCD1 gene under the hEF1α promoter (shown in SEQ ID NO. 3). The specific connection locations and the construction of the lentiviral vector are shown in  FIG. 1 . 
     After lentivirus were packaged, purified and concentrated and used to transfect stem cells, protein expression level of the ABCD1 gene in the stem cells was identified, as shown in  FIG. 2 . Protein expression level identification was performed on the collected HSCs which were transfected by the lentivirus carrying the normal ABCD1 gene to clarify the expression of the ABCD1 gene in HSCs. There was no ABCD1 protein expression in negative control HSCs that were not transfected by the lentivirus, whereas significantly high ABCD1 protein expression was found in the HSCs transfected by the lentivirus carrying the normal ABCD1 gene. The above shows that in the present application, the HSCs can massively express ABCD1 protein by means of the lentivirus, and has great disease treatment potential. The contents without specific techniques or conditions specified in the examples are conducted according to technical conditions described in the literature widely recognized in the art or according to specifications of the corresponding products. The reagents or instruments used herein without manufacturers specified are conventional products commercially available from proper channels. 
     In the present application, a therapeutic procedure for treating ALD disease by using a dual stem cell system obtained through transfection with a lentiviral vector carrying a normal ABCD1 is shown in  FIG. 3 . Stem cells of a patient are mobilized, then peripheral blood of the patient is collected, and hematopoietic stem cells and mesenchymal stem cells in the peripheral blood are isolated. The dual stem cells are transfected by a lentiviral vector carrying a normal ABCD1 gene to obtain stem cells carrying the normal ABCD1 gene. The cells are intravenously injected back into the patient for disease treatment. In clinical trials of autologous transplantation with gene therapy, 7 ALD patients and 3 MLD patients were treated by using the dual stem cells. The treatment process was smooth, the transplantation of the patients was good. The cell processing data is shown in  FIG. 4 . 
     The above are only preferred examples of the present application and are not intended to limit the present application in form or in substance, and for those skilled in the art, various equivalent changes such as variations, modifications and evolutions made in light of the above disclosed contents without departing from the solutions of the present application all are equivalent embodiments of the present application. Any various equivalent changes such as variations, modifications and evolutions made on the above examples in light of the above substantial techniques of the present application are within the scope of the present application. 
     The ASCII text file “Sequence.txt” created on Dec. 30, 2021, having the size of 17.4 kilobytes, is incorporated by reference into the specification.