Dominant positive hnRNP-E1 polypeptide compositions and methods
Inventors
Antony, Asok C. • Tang, Ying-Sheng
Assignees
US Department of Veterans Affairs • Indiana University Bloomington
Publication Number
US-10093706-B2
Publication Date
2018-10-09
Expiration Date
2037-01-30
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Abstract
Described herein are compositions relating to engineered hnRNP-E1 variant polypeptides, nucleic acids encoding such polypeptides, engineered hnRNP-E1 compositions, and methods of use thereof. In some embodiments, the engineered hnRNP-E1 polypeptide contains a C293S substitution and retains the ability to bind to a poly(rC)- and poly(U)-rich 5′-UTR element in its cognate mRNA targets in the absence of homocysteine. In some cases, the engineered hnRNP-E1 compositions provided herein are useful to increase the translation of a subset of mRNAs or to treat certain health conditions as described herein.
Core Innovation
The invention describes engineered hnRNP-E1 variant polypeptides and related compositions, nucleic acids encoding these polypeptides, and methods for their use. These engineered hnRNP-E1 polypeptides include specific amino acid substitutions, such as a C293S substitution, which confers the ability to bind to a poly(rC)/poly(U)-rich cis-element in target mRNAs independently of homocysteine presence. This is based on the finding that point mutations in hnRNP-E1 can mimic homocysteinylated hnRNP-E1, enabling homocysteine-independent binding and modulation of mRNA translation.
The problem addressed arises from cellular folate deficiency, which causes increased intracellular homocysteine leading to homocysteinylation of hnRNP-E1. Homocysteinylated hnRNP-E1 binds to poly(rC)/poly(U)-rich mRNA elements to upregulate translation of relevant proteins, including folate receptor-α. However, controlling hnRNP-E1 binding independently of homocysteine remains challenging, limiting the capacity to modulate expression of these proteins for therapeutic purposes.
This invention solves the problem by creating hnRNP-E1 variants that replicate the high affinity binding of homocysteinylated hnRNP-E1 to target mRNAs without requiring homocysteine. These variant polypeptides can be introduced or expressed in mammalian cells to increase translation of mRNAs with poly(rC)/poly(U)-rich 5′ UTRs. The invention further describes nucleic acids encoding these variants, their formulations in pharmaceutical compositions, and methods for increasing target mRNA translation and treating various health conditions associated with dysregulated protein expression in deficient states.
Claims Coverage
The patent contains multiple independent claims covering variant hnRNP-E1 polypeptides with specific amino acid substitutions, their pharmaceutical compositions, and methods of use for increasing translation of target mRNAs.
Variant hnRNP-E1 polypeptides with cysteine-to-serine substitutions enabling homocysteine-independent RNA binding
Isolated hnRNP-E1 variants comprising at least one amino acid substitution at residues C293, C54, C158, or C201 replaced by serine, with RNA binding portions that bind single-stranded RNA comprising SEQ ID NO:6 in the absence of homocysteine; optionally linked to a protein transduction domain.
Pharmaceutical compositions containing the variant hnRNP-E1 polypeptides
Pharmaceutical compositions comprising the variant hnRNP-E1 polypeptides described above combined with pharmaceutically acceptable carriers suitable for various administration routes.
Methods to increase translation of target mRNAs using the variant hnRNP-E1 polypeptides
Methods of increasing translation of target mRNAs possessing poly(rC)- and poly(U)-rich 5′ untranslated regions in mammalian cells by providing the variant hnRNP-E1 polypeptide, either by administering the protein or by expression within the cell after delivering nucleic acids encoding the polypeptide.
The claims collectively cover engineered hnRNP-E1 polypeptides with specific cysteine-to-serine substitutions that bind RNA independent of homocysteine, pharmaceutical formulations thereof, and methods for enhancing translation of specific target mRNAs in cells, establishing compositions and uses of these variants for therapeutic applications.
Stated Advantages
The engineered hnRNP-E1 variants enable homocysteine-independent binding to target mRNAs facilitating controlled upregulation of translation.
Use of these variants can overcome limitations imposed by physiological homocysteine dependence, allowing modulation of protein expression in various health conditions.
Pharmaceutical compositions enable delivery of these variants or their encoding nucleic acids in vivo or ex vivo for therapeutic benefit.
Documented Applications
Increasing translation of mRNAs encoding folate receptor, hnRNP-E1, collagen alpha (I), human papillomavirus type 16 L2, human herpesvirus 8, erythropoietin, human alpha- and beta-globin, μ-opioid receptor, androgen receptor, p21waf, tyrosine hydroxylase, and neurofilament M.
Treatment of health conditions including human papillomavirus infection, Kaposi's sarcoma, anemia, skin conditions (such as wounds), alpha-thalassemia, beta thalassemia, chronic pain, androgen deficiency, tumors, Parkinson's disease, and nerve injuries.
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