Adeno-associated virus compositions for pah gene transfer and methods of use thereof
An adeno-associated virus and genome technology, applied in the field of adeno-associated virus composition for PAH gene transfer and its application, can solve the problems of insufficient expression duration and insufficient expression level
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Embodiment 1
[0160] Example 1: Human PAH transfer vector
[0161] This example provides human PAH transfer vectors pHMI-hPAH-TC-004, pHMI-hPAH-TC-025, pHMI-hPAH-TC-010, pHMI-hPAH-TC-011 and pHMI-hPAH-TC-012, used Express human PAH in human or mouse cells.
[0162] a) pHMI-hPAH-TC-004
[0163] Such as Figure 1A As shown, the PAH transfer vector pHMI-hPAH-TC-004 contains the following genetic elements from 5' to 3': 5' ITR element, CAG promoter, silently altered human PAH coding sequence, SV40 polyadenylation sequence and 3' ITR element. The sequences of these elements are listed in Table 1. The vector is capable of expressing human PAH protein in cells transduced with the vector, such as human cells or mouse cells.
[0164] Table 1: Genetic elements in the human PAH transfer vector pHMI-hPAH-TC-004
[0165] genetic element SEQ ID NO 5'ITR element 18 CAG promoter 28 Codon-altered human PAH coding sequence 25 SV40 polyadenylation sequence 42 3'...
Embodiment 2
[0185] Example 2: Mouse PAH gene transfer in a mouse model
[0186] This example provides the mouse PAH transfer vector rAAV-CBA-mPAH, which is similar to the human PAH transfer vector pHMI-hPAH-TC-004 described in Example 1, except that the wild-type mouse PAH coding sequence is used to replace the codons Altered human PAH coding sequence. The vector is capable of expressing mouse PAH protein in cells transduced with the vector, such as human cells or mouse cells.
[0187] In short, the Pah - / - (PAH enu2 ) mice were housed in transparent polycarbonate cages with contact pads in isolators. Animals were given Picolab Mouse Diet 5058 ad libitum. Spring or tap water acidified with 1 N HCl to a target pH of 2.5-3.0 was provided ad libitum. Vectors packaged in AAVHSC15 capsids were prepared in PBS (with Ca and Mg) supplemented with 35 mM NaCl, 1% sucrose and 0.05% Pluronic F-68. The formulation was injected intravenously through the tail vein.
[0188] After administration o...
Embodiment 3
[0194] Example 3: Human PAH gene transfer in mouse model
[0195] This example demonstrates that the PAH transfer vector described in Example 1 effectively reverses the phenotype caused by PAH gene deficiency in a mouse model. The mouse model, AAV packaging and formulation, and methods for examining gene transfer efficiency were the same as described in Example 2.
[0196] To examine the efficacy of the five PAH transfer vectors in reversing the phenotype, a single dose of 2.6 × 10 13 vector genome, or 6 × 10 per kg body weight for female mice 13 dose of a vector genome. pah - / - Mice exhibit elevated levels of phenylalanine (Phe) and decreased levels of tyrosine (Tyr) in serum. like Figures 6A-6H As shown, administration of any of the five vehicles resulted in a significant decrease in Phe levels and an increase in Tyr levels within one week. This efficacy persisted for at least 12 weeks in male mice and for at least 6 weeks in female mice. With the exception of pHMI-h...
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