Compositions and methods for treatment of hemopigmentation
A hemoglobin and polynucleotide technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, gene therapy, etc., can solve problems such as loss and complicated application
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Embodiment 1
[0272] Example 1: Expression of HFE protein in hepatocytes from HFE mRNA.
[0273] This example demonstrates that exogenous HFE protein can be produced in cultured primary human hepatocytes following mRNA transfection using a commercially available delivery agent.
[0274] Primary human hepatocytes (Thermo-Fisher Scientific) were purchased, recovered by freezing, and plated according to the manufacturer's recommended protocol. Lipofectamine MessengerMAX (Thermo-Fisher Scientific) was used to transfect codon-optimized HFE-encoding mRNA (modified to use N1-methyl-pseudouridine ["N1MPU"] or 5-methoxyuridine [" 5MOU”] to replace uridine). The RNA sequence used in this example is shown in SEQ ID NO:67, which includes a 5' cap (providing a single 5'A residue), the 5'UTR of SEQ ID NO:33, the HFE coding sequence of SEQ ID NO:4 (encoding the protein of SEQ ID NO:32) and the 3'UTR of SEQ ID NO:35.
[0275] Twenty-four hours after transfection, cells were lysed in RIPA buffer for subs...
Embodiment 2
[0278] Example 2: Duration of HFE protein expression in hepatocytes from HFE mRNA.
[0279] This example demonstrates the duration of exogenous HFE expression following transfection of HFE mRNA using a commercially available delivery agent.
[0280] Primary human hepatocytes (Thermo-Fisher Scientific) were purchased, recovered by freezing, and plated according to the manufacturer's recommended protocol. Lipofectamine MessengerMAX (Thermo-Fisher Scientific) was used to transfect 500 ng of codon-optimized HFE-encoding mRNA (modified to use N1-methyl-pseudouridine ["N1"] or 5-methoxyuridine ["5MU"] replace uridine). The RNA sequence used in this example is shown in SEQ ID NO:67, which includes a 5' cap (providing a single 5'A residue), the 5'UTR of SEQ ID NO:33, the HFE coding sequence of SEQ ID NO:4 ( encodes the protein of SEQ ID NO:32) and the 3'UTR of SEQ ID NO:35.
[0281] Twenty-four hours after transfection (and every day thereafter), cells were lysed in RIPA buffer for...
Embodiment 3
[0284] Example 3: Expression of hepatic HFE protein and reduction of peripheral iron levels in Hfe knockout mice.
[0285] This example demonstrates that administration of HFE-encoding mRNA encapsulated in lipid nanoparticles can produce hepatic HFE protein expression in a dose-dependent manner in Hfe knockout mice.
[0286] In this example, mRNA (SEQ ID NO:67) capable of encoding human HFE was encapsulated in lipid nanoparticles and administered to Hfe via tail vein injection at 0.3 mg / kg, 1 mg / kg and 3 mg / kg. knockout mice. Approximately 48 hours after dosing, livers were harvested and blood was collected for determination of iron levels.
[0287] After a single dose of HFE-encoding mRNA, dose-dependent HFE protein expression was observed in mouse liver homogenates by anti-HFE immunoblotting ( image 3 ). A subsequent restoration of hepcidin gene expression was observed in treated mice by branched DNA (bDNA) assays at levels similar to wild-type controls ( Figure 4 ). ...
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