Application of lentiviral vector EF1? promoter for optimising ABCD1 gene expression to treat adrenoleukodystrophy

Abstract

Provided is a lentiviral vector comprising an EF1? promoter, a normal ABCD1 gene and an NHP/TYF lentiviral vector system, said vector being used for treating adrenoleukodystrophy. The present invention uses transfection into autologous haematopoietic stem cells (HSCs), for ALD gene therapy after being returned, which may be performed in combination with direct intracerebral injection of the lentiviral vector carrying the ABCD1 gene according to the actual circumstances.

Claims

1. A lentiviral vector carrying a normal ABCD1 gene, comprising an ABCD1 gene sequence, a human EF1? promoter sequence and a lentiviral vector NHP/TYF lentiviral vector system, wherein the lentiviral vector comprises the sequence of SEQ ID NO: 3.

2. The lentiviral vector carrying the normal ABCD1 gene according to claim 1, wherein the ABCD1 gene sequence and the human EFla promoter sequence are connected into the lentiviral vector NHP/TYF lentiviral vector system through restriction enzyme sites.

3. A method for preparing a lentiviral vector carrying a normal ABCD1 gene, comprising: connecting an ABCD1 gene sequence and a human EFla promoter sequence into a lentiviral vector NHP/TYF lentiviral vector system through restriction enzyme sites, followed by packaging, purification and concentration, wherein the human EFla promoter sequence haslentiviral vector comprises the sequence of SEQ ID NO: 3.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram illustrating the construction of a lentiviral vector and a production and purification process thereof;

(2) FIG. 2 illustrates protein identification of the protein expression level of the ABCD1 gene in stem cells;

(3) 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

(4) 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

(5) 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.

(6) 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.

(7) 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

(8) A normal ABCD1 gene sequence (shown in SEQ ID NO. 1) and a human EF1? 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 hEF1? promoter (shown in SEQ ID NO. 3). The specific connection locations and the construction of the lentiviral vector are shown in FIG. 1.

(9) 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.

(10) 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.

(11) 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.

(12) 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.