UBE3A gene, expression cassette and application thereof
An expression cassette and open reading frame technology, which can be used in applications, gene therapy, genetic engineering, etc., and can solve problems such as no specific treatment.
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Embodiment 1
[0299] Example 1: Generation of human UBE3A codon-optimized constructs for intracellular expression.
[0300] designed a novel Angelman syndrome therapy that co-delivers the short and long UBE3A isoforms (i.e., human isoform 1 (also referred to as "short") and human isoform 3 at appropriate expression ratios (also referred to as "long")), referred to herein as PHP.B / SYN-UBE3A, which comprises the open reading frame (ORF) hUBE3A_isoform 1 >>3. This is an important technical advantage, since viral vector packaging constraints usually only allow the expression of one isoform. This construct overcomes two major challenges: (1) delivering UBE3A broadly throughout the brain; and (2) avoiding UBE3A overexpression that may be associated with a syndromic form of autism (Dup15q syndrome). AAV9-derived capsids, when delivered early in life, can broadly transduce neurons throughout the brain. While there are other AAV capsids that may enable broader gene transfer in the brains of nonhum...
Embodiment 2
[0306] Example 2: Human UBE3A codon-optimized construct treatment to restore anatomical and behavioral phenotypes in AS model mice.
[0307] Figure 9 showed that PHP.B / hUBE3A was generally well tolerated in both WT and AS model mice, as demonstrated by gene therapy-treated groups (WT+AAV and AS+AAV) and vehicle-treated WT controls (WT+vehicle) in Equal body weight at one month of age to demonstrate. Vehicle-treated AS mice gained excess body weight as adults, consistent with previous reports (Judson et al., JNeurosci, 2017:37(31):7347-7361) and clinical observations of adult obesity in AS individuals (Bindels- de Heus et al., Am J Med Genet A, 2020:182(1):53-63). In the AS+AAV group, PHP.B / hUBE3A treatment completely rescued this phenotype.
[0308] The ability of neonatal ICV PHP.B / hUBE3A treatment to rescue hyperpermeable and reproducible AS mouse model defects was further tested ( Figure 10 Panel A; Rotaru et al., Neuroscience, 2020: Epub ahead of print). Figure 10 Pa...
Embodiment 3
[0313] Example 3: Construct therapy of humanized cells in vitro and in vivo.
[0314] The constructs described in Example 1 were delivered in vitro to patient-derived iPSCs that had differentiated into neurons and exhibited an AS cell phenotype. The recipient cells are then observed for reversal of the characteristic AS cell phenotype compared to appropriate control cells that have not received the construct.
[0315] AS iPSC lines can be generated from peripheral skin or blood cells of patients, and their pluripotency is conferred by retroviral or lentiviral vectors expressing key transcription factors OCT4, SOX2, KLF4, MYC, and LIN28 (Takahashi et al., Cell, 2007:131:861 -872; Sommer et al., Stem Cells, 2008:27:543-549). In certain preparations, CRISPR / Cas9 editing can be used to introduce AS that results in loss of UBE3A into iPSCs from neurotypical patients, resulting in new AS-iPSC lines accompanied by isogenic controls. ASiPSCs can be differentiated into neurons using ...
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