Methods and compositions for increasing protein expression and/or treating a haploinsufficiency disorder
Inventors
Coller, Jeffery M. • Sweet, Thomas • Lodish, Harvey
Assignees
Case Western Reserve University • Whitehead Institute for Biomedical Research
Publication Number
US-12281314-B2
Publication Date
2025-04-22
Expiration Date
2039-09-26
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Abstract
A tRNA that hybridizes to a non-optimal codon can be used to increase expression in a mammalian cell of a gene product encoded by a gene containing the non-optimal codon or to treat a haploinsufficiency disorder in a subject having a haploinsufficient gene containing the non-optimal codon.
Core Innovation
This invention describes methods and compositions for increasing the expression of a gene product encoded by a gene containing one or more non-optimal codons in mammalian cells, and for treating haploinsufficiency disorders. The core approach involves introducing exogenous transfer RNAs (tRNAs) that hybridize specifically to these non-optimal codons, where such tRNAs are less abundant in cells, thereby enhancing mRNA stability and gene product output.
The problem addressed is that certain gene mutations or codon usage result in diminished mRNA stability and insufficient production of functional proteins, leading to disorders such as haploinsufficiency. By targeting the specific non-optimal codons (e.g., ATA, GTA, AGA) with their cognate tRNAs, the methods described facilitate increased translation efficiency and stability of the mRNA transcript, improving protein expression.
The invention further provides that administration of these exogenous tRNAs or expression vectors encoding these tRNAs can increase expression selectively for the target gene product without substantially affecting other gene products in the cell. This strategy applies whether the tRNAs are delivered directly or expressed from introduced genetic constructs, and can be used regardless of the specific nature of the inactivating mutation causing haploinsufficiency.
Claims Coverage
The claims define two central inventive features regarding exogenous tRNA introduction and expression vector approaches for enhancing gene product expression linked to non-optimal codons.
Introduction of exogenous tRNAs targeting non-optimal codons
The method involves introducing into a mammalian cell an effective amount of three exogenous transfer RNAs (tRNAs), each comprising an anticodon that hybridizes to a first, second, and third non-optimal codon (where at least one is selected from ATA, GTA, and AGA). Each tRNA must be capable of aminoacylation with an amino acid. This introduction leads to increased expression of the gene product relative to a similar cell lacking these tRNAs.
Combination of expression vectors encoding tRNAs for non-optimal codons
This method includes introducing into a mammalian cell an effective amount of a combination of three expression vectors, each capable of expressing an exogenous tRNA with an anticodon that hybridizes to a first, second, or third non-optimal codon (again, at least one of which is ATA, GTA, or AGA). Upon expression, these tRNAs are capable of being aminoacylated and increase the gene product expression relative to a cell without the introduced vectors. The vectors may be the same or different and may include viral or adeno-associated virus (AAV) vectors, encompassing sequences such as SEQ ID NO: 4–9.
Thus, the inventive features protect both direct tRNA introduction and vector-mediated tRNA expression for augmenting protein production from genes containing specified non-optimal codons, covering a range of delivery strategies and genetic targets.
Stated Advantages
The methods allow increased expression of gene products from genes containing non-optimal codons, which can lead to at least 7-fold increases in target mRNA and improved protein output.
The approach can selectively enhance expression of the target gene product without substantially affecting other gene products present in the cell.
The methods and compositions can treat haploinsufficiency disorders regardless of the mutation or mechanism by which an allele was inactivated.
Transfer RNAs are relatively small and can be produced recombinantly, rendering the approach broadly accessible and adaptable.
Documented Applications
Treatment of haploinsufficiency disorders, including but not limited to Dravet syndrome, 5q-syndrome, CHARGE syndrome, Smith-Magenis syndrome, Rett syndrome (congenital variant), Sotos syndrome, mental retardation syndromes, and various others as listed in the patent's tables.
Increasing expression of gene products for a comprehensive set of genes associated with haploinsufficiency, including SCN1A, and other genes such as AGGF1, ARHGAP31, BMPR2, CHD7, COL2A1, COL3A1, and additional genes explicitly stated in the document.
Use in combination with approved epilepsy therapies such as DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), or valproic acid for the treatment of Dravet syndrome.
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