SEQUENCE LISTING

The instant application contains a Sequence Listing, created on Jul. 9, 2020; the file, in ASCII format, is designated H1789760.txt and is 65.9 KB in size. The file is hereby incorporated by reference in its entirety into the instant application.

BACKGROUND

Obesity is a global epidemic that plagues the human society, threatening the health of both adult and children. Effective pharmacological therapies for obesity are urgently needed. Obesity-related pathologies include, among others, diabetes and liver disease. Adipose tissue overgrowth is the root of obesity, with deleterious health effects. Adipose tissue is composed of white and brown adipose tissue (BAT). White adipose tissue (WAT) stores triglycerides in adipocytes, and BAT burns triglycerides and glucose for generating heat. The development of obesity depends not only on the balance between food intake and caloric utilization but also on the balance between BAT and WAT. Higher BAT is correlated with leanness in the adult and greater muscle volume in children, indicating that functional BAT benefits both energy homeostasis and muscle growth. In humans, BAT is abundant in infants, and decreases with age. Recently, the discovery of functional BAT in adult individuals raised the possibility of treating obesity by activating BAT. However, compositions and methods for increasing BAT are lacking. The present disclosure is directed to overcoming these and other deficiencies in the art.

SUMMARY

The following disclosure includes improvements over such shortcomings.

In an aspect, provided is a polynucleotide, including a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein, wherein the cis-regulatory element includes an uncoupling protein 1 enhancer and an uncoupling protein 1 promoter. In an example, the uncoupling protein 1 enhancer has at least 90% identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO 4, and SEQ ID NO: 7. In another example, the uncoupling protein 1 enhancer is selected from SEQ ID NO: 1, SEQ ID NO 4, and SEQ ID NO: 7. In another example, the uncoupling protein 1 promotor has at least 90% identity with a sequence selected from SEQ ID NO: 2, SEQ ID NO 5, and SEQ ID NO: 8. In still another example, the uncoupling protein 1 promotor is selected from SEQ ID NO: 2, SEQ ID NO 5, and SEQ ID NO: 8. In yet another example, the cis-regulatory element has at least 90% homology with a sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9. In a further example, the cis-regulatory element is selected from SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.

In another example, the vestigial like 4 protein has at least 90% homology with a sequence selected from SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33. In still another example, the vestigial like 4 protein is selected from SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33. In yet another example, the sequence encoding a vestigial like 4 protein has at least 90% identity with a sequence selected from SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32. In a further example, the sequence encoding a vestigial like 4 protein is selected from SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32.

In another example, the vestigial like 4 protein has from 0 to 3 substitutions to a sequence selected from SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, wherein the substitutions are not in a TDU domain. In still another example, the vestigial like 4 protein has from 0 to 3 substitutions to a sequence selected from SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33, wherein the substitutions are not in a TDU domain.

Another example further includes an intron between the cis-regulatory element and the nucleotide sequence encoding a vestigial like 4 protein. In another example, the intron has at least 90% homology with SEQ ID NO: 34. In still another example, the intron is SEQ ID NO: 34.

Another example includes a nucleotide sequence having at least 90% homology with SEQ ID NO: 35. An example includes a nucleotide sequence of SEQ ID NO: 35. Another example includes a nucleotide sequence having at least 90% homology with SEQ ID NO: 36. An example includes a nucleotide sequence of SEQ ID NO: 36.

Another example further includes a nucleotide sequence encoding a reporter protein. In another example, the reporter protein is selected from a green fluorescent protein, a yellow fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a luciferase protein, a beta-galactosidase protein, a glutathione S-transferase protein, a chloramphenicol acetyltransferase protein, and any combination of two or more of the foregoing. In still another example, the reporter protein includes a green fluorescent protein. In yet another example, the reporter protein includes SEQ ID NO: 37. In a further example, the nucleotide sequence encoding a reporter protein includes SEQ ID NO: 38.

Another example includes a nucleotide sequence having at least 90% homology with SEQ ID NO: 39. An example includes SEQ ID NO: 39. Another example includes a nucleotide sequence having at least 90% homology with SEQ ID NO: 40. An example includes SEQ ID NO: 40.

In another aspect, provided is a viral vector including any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein. In an example, the viral vector includes an adenoviral associated vector.

In another aspect, provided is a cell transfected with any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein. In an example, the cell was contacted with any of the foregoing examples of a viral vector that include any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein.

In still another aspect, provided is an organism transfected with any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein. In an example, the organism was contacted with any of the foregoing examples of a viral vector.

In another aspect, provided is a method. In an example, the method includes transfecting a cell with any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein. In another example, transfecting includes contacting the cell with any of the foregoing examples of a viral vector that include any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein. In still another example includes transfecting an organism with any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein. In yet another example, transfecting includes contacting the organism with any of the foregoing examples of a viral vector that include any of the foregoing examples of a polynucleotide that include a cis-regulatory element and a nucleotide sequence encoding a vestigial like 4 protein.

In another example, the organism is a mammal. In still another example, the organism is a human.

In an example of the method, the vestigial like protein 4 does not include an HF to AA substitution in a TDU domain of the vestigial like protein 4 and the transfecting includes increasing a ratio of a volume of brown adipose tissue to a volume of white adipose tissue in the organism. In still another example, the vestigial like protein 4 does not include an HF to AA substitution in a TDU domain of the vestigial like protein 4 and the transfecting includes increasing a volume of brown adipose tissue in the organism, decreasing the volume of white adipose tissue in the organism, or both. In yet another example, the vestigial like protein 4 does not comprise an HF to AA substitution in a TDU domain and the transfecting includes reducing a ratio of a volume of adipose tissue to a volume of non-adipose tissue in the organism.

In a further example, the vestigial like protein 4 does not include an HF to AA substitution in a TDU domain, and the organism is obese or is at risk of developing obesity. In still a further example, the vestigial like protein 4 does not include an HF to AA substitution in a TDU domain, and the transfecting includes preventing obesity in the organism. In yet another example, the vestigial like protein 4 does not include an HF to AA substitution in a TDU domain, and the transfecting includes treating obesity in the organism. In another example, the vestigial like protein 4 does not include an HF to AA substitution in a TDU domain, and the transfecting includes reducing obesity in the organism.

In an example of the method, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, wherein the transfecting includes reducing a volume of adipose tissue of the organism. In another example, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, and the transfecting includes reducing a volume of brown adipose tissue of the organism. In still another example, the vestigial like protein 4 includes an HF to AA substitution in a TDU domain, and the organism is obese or is at risk of developing obesity. In yet another example, the vestigial like protein 4 includes an HF to AA substitution in a TDU domain, and the transfecting includes preventing obesity in the organism. In a further example, the transfecting includes treating obesity in the organism. In still a further example, the vestigial like protein 4 includes an HF to AA substitution in a TDU domain, and the transfecting includes reducing obesity in the organism.

In an example of the method, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, and the transfecting includes reducing fatty acid synthesis in the organism. In another example, the vestigial like protein 4 includes an HF to AA substitution in a TDU domain, and the organism has hepatic steatosis or is at risk for developing hepatic steatosis. In still another example, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, and the transfecting includes preventing hepatic steatosis in the organism. In yet another example, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, and the transfecting includes treating hepatic steatosis in the organism. In a further example, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, and the transfecting includes reducing hepatic steatosis in the organism.

In an example of the method, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, and the organism has diabetes or is at risk of developing diabetes. In another example, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, wherein the transfecting includes preventing diabetes in the organism. In still another example, the vestigial like protein 4 includes an HF to AA substitution in each of two TDU domains, wherein the transfecting includes treating diabetes in the organism.

