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The safety and health benefits of the expressed genes together with the low-risk profile of AAV-mediated gene delivery yield an approach that may avoid the risk of negative, off-target effects associated with small molecule therapies. Crucially, we have also demonstrated that individual longevity gene therapies can be easily combined into a single therapeutic mixture. This serves as an alternative to the traditional therapeutic approaches that, when testostetone treating multiple diseases, require multiple interventions with unrelated substances, which in turn, increase the accumulative exposure to negative side effects.

A single-dose combination Testostwrone therapy may also help alleviate issues associated with immune response when considering the alternative of multiple independent AAV-delivered therapies.

Future studies may build on the combination AAV therapy concept presented here to treat the many diseases of aging and perhaps, also as a means ranhe address testosterone range normal process of aging itself. The AAV was created using triple transfection of HEK293T cells and iodixanol gradient purification as described previously. The helper plasmid, capsid, and gene of interest testosterone range normal terminal repeat plasmid) were transfected at a 2:1:1 molar ratio.

The media and cells were collected 72 h posttransfection. Wheat germ agglutinin (WGA) was from ABCAM 20528, and DAPI was from SIGMA. Samples were formalin fixed for 24 h and then, paraffin embedded.

The animals were in Harvard Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities. Filtered tap water, acidified to a pH of 2. Testosterone range normal the UUO experiments, mice were purchased at testosterone range normal wk old (JAX tesotsterone 000664).

For the obesity tesotsterone diabetes testoserone, mice were purchased at 17 wk old (JAX stock 380050). All mouse protocols were approved by the Harvard Institutional Animal Care and Use Committee (IACUC) and Committee on Biological Safety (COMS) committees. The AAV was delivered via retroorbital injections.

All mice were randomized based on weight for each experiment. The mice were fasted overnight for testosterpne h for the oral glucose tolerance test (OGTT) and the pyruvate tolerance test (PTT). The mice were only fasted for 6 h for the insulin tolerance test (ITT). These were performed as previously described. The blood glucose was measured at 15- 30- testosterpne, and 120-min intervals.

A One Touch Ultra glucose monitor and black strips were used. Mice were positioned with the testosyerone and neck fully extended to ensure a patent airway. UUO was achieved by exposing the left kidney through the left flank.

The ureter was obstructed completely near the renal pelvis using boobs pregnant 4-0 silk braided polyester Normak tie at 2 points. Testosterone range normal skin was stapled using sterile staples.

Cross bayer sutures were used. The mice were then euthanized at 7 or 14 testosterone range normal postsurgery, and tissues and norml were collected for analysis. The mice were randomized and blinded from the surgeons such that they did not know which mice received which therapy.

Aortic constriction was induced in adult mice through constriction of testosterone range normal aorta. An incision was made in the chest wall at the third intercostal space. A rodent rib spreader was inserted, and the ribs were gently spread to allow access to the thoracic cavity. A blunted 26-gauge needle was placed on top of the aorta, and ligation was tied around the needle. The needle was then carefully removed from under the tie.

The rib spreader was then removed, and the lung was reinflated. The ribs, chest musculature, and skin were closed using sterile 5. Sham-operated animals underwent similar procedures without constriction of the aorta. Animals were testosterone range normal monitored until full recovery from anesthesia. After the animal had testosteerone testosterone range normal (and was able to protect its airway), the animal testosterone range normal extubated.

The surgeon minimized postoperative pneumothorax by placing the final thoracotomy rib-closing suture when the lungs were expanded.

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Comments:

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