Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies

Track A Basic Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138319/1/jia218438.pd

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach.

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre's enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine

Comparison of 2 doses of recombinant human thyrotropin for thyroid function testing in healthy and suspected hypothyroid dogs.

BACKGROUND: Various protocols using different doses of recombinant human thyrotropin (rhTSH) in TSH stimulation testing have been described. However, the influence of TSH dosage on thyroxine (T4) concentration has not yet been evaluated in suspected hypothyroid dogs. OBJECTIVE: To evaluate the effectiveness of 2 doses of rhTSH. ANIMALS: Fifteen dogs with clinical signs consistent with hypothyroidism and abnormal stimulation results with 75 microg rhTSH and 18 clinically healthy dogs. METHODS: All dogs were stimulated with 75 and 150 microg rhTSH IV in a 1st and 2nd stimulation test, respectively. Blood samples were taken before and 6 hours after rhTSH administration for determination of total T4 concentration. RESULTS: Using the higher dose led to a normal test interpretation in 9 of the 15 dogs, in which stimulation had been abnormal using the lower dose. Based on follow-up information, hypothyroidism was excluded in 7 of these 9 dogs. In all 6 dogs with a blunted response to the higher dose, hypothyroidism could be confirmed. Healthy dogs showed significantly higher post-TSH T4 concentrations with the higher compared with the lower dose. Post-TSH T4 concentrations after TSH stimulation were not related to dogs' body weight in either healthy or diseased dogs. CONCLUSIONS AND CLINICAL RELEVANCE: TSH dose significantly influenced test interpretation in suspected hypothyroid dogs. Differentiation between primary hypothyroidism and nonthyroidal disease was improved with 150 microg rhTSH. Because this effect was independent of the dogs' body weight, the higher dose is recommended in dogs that have concurrent disease or are receiving medication

Thyroid enlargement and its relationship to clinicopathological parameters and T(4) status in suspected hyperthyroid cats

To relate thyroid size to routine blood parameters and T(4) status the ventral neck of 161 cats with clinical signs consistent with hyperthyroidism was examined by two independent observers using a semi-quantitative palpation system. Thyroid gland size of each side was scored from 0 (non-palpable) to a maximum of 6 (>25mm). In 127 of the 161 cats, at least one thyroid gland was palpable. The palpation score was significantly correlated with the T(4) concentration. The 17 hyperthyroid cats had significantly higher palpation scores than the 110 euthyroid cats. Euthyroid animals with a palpation score >/=3 were significantly older, had higher body weights, lower alkaline phosphatase, alanine aminotransferase, phosphate, and urine specific gravity, but higher lipase and creatinine concentrations than hyperthyroid cats. Our study demonstrates that although no reliable conclusion on the functional status of the thyroid can be drawn based on its size the likelihood of hyperthyroidism increases with increasing size of the gland

Primärer Hyperaldosteronismus bei Katzen

Primary hyperaldosteronism is a clinical syndrome characterized by an elevated aldosterone secretion by the adrenals. The present case series describes 7 cats with primary hyperaldosteronism, which were presented between 2002 and 2011. Common clinical symptoms were weakness, anorexia, cervical ventroflexion and blindness. All cats showed hypokalemia. In 6 cats, blood pressure was determined: 5 cats showed hypertension, of which 4 animals exhibited retinal detachment and blindness. In the ultrasonographic examination, unilateral adrenomegaly was present in 6 cats whereas one animal showed normal adrenals. In 4 cats, the serum aldosterone concentration was above the reference range. Five cats underwent unilateral adrenalectomy, which was accomplished uneventfully and returned the electrolytes back to normal. Histopathological examination of the adrenals revealed 2 carcinomas and 4 adenomas; one cat with ultrasonographic normal adrenals exhibited bilateral nodular hyperplasia

Primary hyperaldosteronism in cats: a series of seven cases

Primary hyperaldosteronism is a disease characterised by elevated aldosterone secretion by the adrenal glands. The present case series describes seven cats with primary hyperaldosteronism presented between 2002 and 2011. Common clinical signs were weakness, anorexia, cervical ventroflexion and blindness. All cats showed hypokalaemia. Blood pressure measurement in six cats revealed hypertension in five; four of the five cats were blind because of retinal detachment. Ultrasonographic examination showed unilateral adrenomegaly in six cats and normal adrenals in one. The serum aldosterone concentration exceeded the reference range in four cats. Five cats underwent unilateral adrenalectomy, which was without complications and led to normalisation of the electrolyte concentrations. Histological examination of the adrenal glands revealed adrenocortical adenoma in four cats and adrenocortical carcinoma in two; the cat with ultrasonographic normal adrenals had bilateral nodular adrenal hyperplasia