AAV capsid design: A Goldilocks challenge.

In vivo therapeutic gene transfer has emerged as a novel class of medicines. Its feasibility relies on the safe and efficacious delivery of genetic cargo to the appropriate targets. The adeno-associated virus (AAV) vector manifested itself as a preferred gene delivery vehicle enabling therapeutic gene expression for several clinical indications. Here, we cover the recent trends in AAV capsid engineering to enhance its targeting specificity, safety, and endurance. While each and every desirable trait can be individually remodeled, combining several attributes in one capsid amounts to a significant engineering challenge. Taking advantage of virion structure and phylogenetics, harnessing directed evolution, sequence analyses, and machine learning, researchers develop novel capsid variants to realize the goals of safe and enduring gene therapy.

[Experience of organ spare sialolitiasis treatment using sialoendoscopy]. / Opyt organosberegayushchego khirurgicheskogo lecheniya sialolitiaza s ispol'zovaniem sialoskopii.

The aim of the study was to evaluate the effectiveness of organ spare sialolitiasis treatment by sialoscopy-guided techniques. The study included 317 patients with parotid and submandibular sialolitiasis. In 22 patients conventional surgical approach was used because of distal location and larger size of concrements, while in 295 miniinvasive sialoscopy-guided procedures were performed. The use of sialoscopy-guided approach drastically diminished the rate of submandibular glands extirpations (performed in 5% of patients), as well as early and long-term surgical complications.

Hypothalamus Specific Re-Introduction of SNORD116 into Otherwise Snord116 Deficient Mice Increased Energy Expenditure.

The Snord116 gene cluster has been recognised as a critical contributor to the Prader-Willi syndrome (PWS), with mice lacking Snord116 displaying many classical PWS phenotypes, including low postnatal body weight, reduced bone mass and increased food intake. However, these mice do not develop obesity as a result of increased energy expenditure. To understand the physiological function of SNORD116 better and potentially rescue the altered metabolism of Snord116-/- mice, we used an adeno-associated viral (AAV) approach to reintroduce the product of the Snord116 gene into the hypothalamus in Snord116-/- mice at different ages. The results obtained show that mid-hypothalamic re-introduction of SNORD116 in 6-week-old Snord116-/- mice leads to significantly reduced body weight and weight gain, which is associated with elevated energy expenditure. Importantly, when the intervention targets other areas such as the anterior region of the hypothalamus or the reintroduction occurs in older mice, the positive effects on energy expenditure are diminished. These data indicate that the metabolic symptoms of PWS develop gradually and the Snord116 gene plays a critical role during this process. Furthermore, when we investigated the consequences of SNORD116 re-introduction under conditions of thermoneutrality where the mild cold stress influences are avoided, we also observed a significant increase in energy expenditure. In conclusion, the rescue of mid-hypothalamic Snord116 deficiency in young Snord116 germline deletion mice increases energy expenditure, providing fundamental information contributing to potential virus-mediated genetic therapy in PWS.

An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina.

Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1ß, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1ß was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1ß. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1ß.

[Immediate results of complex treatment of resectable rectal neoplasms of distal localization].

The impact of neoadjuvant chemoradiation on immediate results of treatment of resectable cancer recti, using large-fractionized radiation in combination with endolymphatic chemotherapy, was estimated. Using the method proposed 64 patients were treated (the main group). In control groups there were included 63 patients, to whom a course of a large-fractionized radiation on background of intravenous chemotherapy was applied, and in 91 patients a large-fractionized radiation only was used. The intraoperative complications rate in the main and control groups have had constituted, accordingly, 16, 6.3 and 3.3%. Postoperative complications have had occurred in 12.5% of patients in the main group, and in 15.9% and 14.3% - in the control groups. The abscesses formation was noted in a small pelvis cavity in 4.7% patients of the main, and in 4.8 and 4.4% - in the control groups. Necrosis of the descended gut was revealed in 10 (4.6%).

[Neoadjuvant chemotherapy and radiation therapy of resectable cancer recti of distal localization].

The method of combined neoadjuvant treatment of resectable cancer recti, consisting of preoperative radiaton therapy, using big-fractionized intensive irradiation on the endolymphatic chemotherapy background together with fluorouracil with following surgical intervention (main group), in terms up to 72 h, was elaborated in the clinic. The patients, to whom the chemotherapy and radiation therapy were conducted, were included into control groups. Postoperative complications have had occurred in 8 (12.5%) patients of the main group and in 10 (15.87%) and 13 (14.29%)--in control groups. The five-year survival indices in the main group have constituted (73.5 +/- 6.3)%, and in control groups--(64.6 +/- 5.8) and (64.4 +/- 6.8)%. The local recurrence rate in the main group have constituted (6.2 +/- 3.0)%, and of the remote metastatizing--(15.6 +/- 4.5)%.

[The two-staged intervention for complicated colonic cancer].

The methods of colonic resection and a two-barrel colostomy closure, after performance of modified Mikulicz operation for complicated colonic cancer with formation of a two-staged extraperitoneal invagination duplicature anastomosis, were proposed. Postoperative complicationshave had occurred in 10 (10.9%) patients, including the anastomotic sutures insufficiency--in 2 (2.2%).

[Intraoperative rupture of cerebral aneurysm--incidence and risk factors].

OBJECTIVE: To reveal and assess risk factors for intraoperative rupture (IOA) of aneurysms, which will reduce the incidence of this complication and improve the outcome of treatment. METHODS: Direct surgical interventions made in 610 cerebral aneurysms at the Vascular Department of N.N. Burdenko Research Institute of Neurosurgery, Russian Academy of Medical Sciences, in 2002 to 2005 were analyzed in 519 patients. Under study were the following factors: onset of IOA; site and anatomic and topographic features of aneurysms; time of an operation (acute or cold subarachnoidal hemorrhage), presence or absence of hemorrhage in the history, rate and severity of hemorrhage (according to the Hunt-Hess classification), temporary clipping of the nutrient arteries. RESULTS: The incidence of IOR was 11.7% of the operated aneurysms and 13.7% of the patients. Ruptures occurred during aneurysmal manipulations in 96% of cases. Moderate and significant hemorrhages requiring a surgeon to make additional measures to improve them more frequently occurred in IOR. The highest rate of IOR was recorded at surgery for aneurysms of the anterior cerebral artery--the anterior communicating artery. The incidence of ruptures increased with their greater sizes. There was a significant increase in the incidence of IOR in aneurysms with diverticula (p = 0.004) and with wide necks (p = 0.04). The level of IOR in the patients operated on within the first 3 weeks after SAK was 3 times higher than in those operated on in the cold period (p < 0.05). The incidence of IOR was rather low in the treatment of asymptomatic aneurysms (2.5%). The risk for IOR was higher in patients with recurring hemorrhage. Preventive temporary occlusion substantially lessens the risk of IOR. CONCLUSION: The low rate of IOA in the treatment of asymptomatic aneurysms is another argument in favor of the development of surgery for unruptured asymptomatic aneurysms. Timely temporary clipping of the nutrient artery at surgery substantially reduces the risk of IOR in all cases.

[Intraoperative contact Doppler ultrasonography in the surgery of cerebral aneurysm].

Vascular occlusion leading to the development of an ischemic focus or incomplete aneurysm closure is one of the main causes that deteriorate the total results of surgical treatment in patients with cerebral aneurysms. The study covering 50 patients with 54 aneurysms who were evaluated for vascular patency by contact Doppler ultrasonography (CDUS) assessed the role and reliability of this technique. In the study group, the ratio of males to females was 30:24, their mean age was 44 years (23-67 years); the ratio of patients with acute subarachnoidal hemorrhage (SAH) to those without SAH was 24:30. The functional outcomes were assessed by a modified Rankin scale and compared with those in the control group. The clip site was inspected by the results of CDUS in 19 (31.7%) cases; clip reposition was made in 16 (26.7%) cases due to vascular stenosis; an additional clip was use to exclude the aneurysmal functioning part in 1 (1.7%) case. Vascular stenosis was detected in 2 (3.3%) cases; however, the position of clips remained unchanged since aneurysm was excluded under the conditions of its rupture. According to the modified Rankin scale, good results were slightly more in the study group of "acute" patients than in the control group: 96.7% versus 90%; the similar results were obtained in non-SAH patients: 66.7% versus 62.5%; lambda < 1.36). CDUS is a noninvasive, simple, reliable, and inexpensive technique for blood flow evaluation.

Production of recombinant adeno-associated virus vectors.

Recombinant adeno-associated virus (rAAV) is a prototypical gene therapy vector characterized by excellent safety profiles, wide host range, and the ability to transduce differentiated cells. Numerous rAAV-based vectors providing efficient and sustained expression of transgenes in target tissues have been developed for preclinical studies. Interest in rAAV has been driven by advances in production methods originally developed for rAAV serotype 2 vectors and expanded to include alternative serotypes. The transition to clinical trials is dependent on the development of scalable production methods of Good Manufacturing Practice-grade vectors described in this review.

[Immediate results of transabdominal rectal resection with the duplicature anastomosis formation].

There were presented immediate results of surgical treatment of 425 patients with the upper ampullar and rectosygmoid portion cancer recti (CR), to whom in 1989-2002 transabdominal rectal resection, using the duplicature anastomosis, was performed. In 93.4% of observations, according to histological investigation data, adenocarcinoma was diagnosed. Concurrent diseases were revealed in 226 (53.2%) patients. The technique of transabdominal CR resection is depicted. There were noted fair immediate results in treatment of the patients: postoperative complications rate have constituted 13.2%, postoperative mortality--2.6%. In structure of postoperative complications purulent-septic one had prevailed, similarly were complications with cardiovascular and respiratory systems affections.

[Remote results of the treatment of proximal rectal cancer].

There were presented the late results of surgical treatment in 425 patients, suffering upper ampullar and rectosygmoid rectal cancers, to whom transabdominal rectal resection with the duplicature anastomosis formation in 1989--2002 yrs was done. In 93.4% of observations, according to histological investigation data, adenocarcinoma was diagnosed. Concurrent diseases were noted in 226 (53.2%) of patients. The postoperative complications rate was 13.2%, postoperative mortality--2.6%. Late result of treatment was fair.

[Surgical treatment for asymptomatic non-ruptured intracranial aneurysms]. / Khirurgicheskoe lechenie asimptomnykh nerazorvavshikhsia intrakranial'nykh anevrizm.

The study was undertaken to analyze the outcomes of treatment in 37 patients with 44 non-ruptured aneurysms (NRA) at different sites. The paper presents a detailed classification of NRA, identifies individuals at risk for aneurysmal disease in whom an active search for aneurysms until they rupture is recommended to prevent aneurysmal subarachnoidal hemorrhages and their sequels. Total aneurysmal exclusion was achieved in 93.2% of the cases; the aneurysmal walls were consolidated with surgical gauze in 7.2%. There were no fatal cases. Postoperative progression of focal neurological symptoms was noted in 4 patients. In 3 patients, these symptoms were entirely reversible. All NRAs should be operated on irrespective of their size particularly in young and middle-aged patients since this is technically possible and there are no concomitant diseases that make surgery contraindicated. It is expedient to perform computed or magnetic resonance imaging in combination with angiography in risk-group patients in order to reveal or exclude aneurysm.

Differential modulation of energy balance by leptin, ciliary neurotrophic factor, and leukemia inhibitory factor gene delivery: microarray deoxyribonucleic acid-chip analysis of gene expression.

Most obese animal models, whether associated with genetic, diet-induced, or age-related obesity, display pronounced leptin resistance, rendering leptin supplement therapy ineffective in treating obesity. Ciliary neurotrophic factor (CNTF) has been recently used to invoke leptin-like signaling pathways, thereby circumventing leptin resistance. In the current study, we characterize immediate and long-term molecular events in the hypothalamus of rats exposed to the sustained ectopic expression of leptin, CNTF, or leukemia inhibitory factor, another neurocytokine of IL-6 family, all delivered centrally via a viral vector. The respective transgene-encoded ligands induced similar but not identical metabolic responses as assessed by the reduction in body weight gain and changes in food intake. To define molecular mechanisms of weight-reducing and anorexigenic action of cytokines, we have analyzed the gene expression profiles of 1300 brain-specific genes in the hypothalami of normal rats subjected to the prolonged cytokine action for 10 wk. We present evidence that constitutive expression of cytokines in the brain induces changes in gene expression characteristic of chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (leukemia inhibitory factor). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.

Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units.

Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology.

Hypothalamic delivery of doxycycline-inducible leptin gene allows for reversible transgene expression and physiological responses.

Our purpose was to incorporate regulation into the recombinant adeno-associated virus encoding leptin by introducing a tet-inducible promotor. This system, TET-Ob, allows for control of leptin gene expression via doxycycline in drinking water. F344XBN rats (aged 4 months) were given a hypothalamic injection of TET-Ob or control virus. During 34 days of doxycycline (doxy) administration to all rats (STAGE 1), TET-Ob rats gained 50.7% less mass, ate 10.4% less food, and had a 77.5% reduction in serum leptin as compared with controls. Doxy was then withdrawn from half of the TET-Ob rats for 32 days (TET-Ob-OFF), while half continued to receive doxy (TET-Ob-ON) (stage 2). During stage 2, TET-Ob-ON rats gained 44.8% less mass than TET-Ob-OFF and ate significantly less food than both TET-Ob-OFF and controls. Serum leptin increased to 83.4% of control values in TET-Ob-OFF, but remained very low in the in TET-Ob-ON. At death, visceral adiposity was 14.5% of controls in TET-Ob-ON animals, but had risen to 76.9% of controls in TET-Ob-OFF. A reversible increase in both leptin signal transduction in the hypothalamus and uncoupling protein expression in brown adipose was recorded. This system allows for more precise regulation of gene therapy-mediated fat loss.

Peripheral but not central leptin prevents the immunosuppression associated with hypoleptinemia in rats.

Leptin is a peripheral immunoenhancing reagent that directly activates splenic lymphocytes in mice. We found that a 48 h fast in rats resulted in a decrease in serum leptin that was accompanied by a lower delayed-type hypersensitivity (DTH) response. Peripheral leptin replacement completely restored this response in fasted animals. We employed a recombinant adeno-associated virus (rAAV) system to deliver leptin gene directly into rat brain to assess the effect of sustained long-term central expression of leptin on immune responses. The rAAV-leptin rats had elevated central leptin over the 60 day duration of the experiment, whereas body fat and circulating leptin fell to near zero levels. The DTH response was significantly reduced by 10-20% in rats receiving rAAV-leptin compared with the control rats, and the difference was maintained for over 50 h. When the rats undergoing rAAV-leptin gene therapy were given either murine recombinant leptin or PBS s.c., rats receiving leptin had a 17% higher DTH response than rats receiving PBS. The isolated splenocytes from the former group also proliferated 34% more in vitro in response to the mitogen concanavalin A as compared with the latter group. These results suggest that peripheral leptin has a dominant role in maintaining T-cell-mediated immune responses in rats, and central leptin is unable to compensate for the immunosuppression associated with peripheral hypoleptinemia. Furthermore, preservation of normal cell-mediated immune responses does not require fat tissue as along as serum leptin levels are maintained.

Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats.

The purpose of this study was to determine if long-term leptin treatment desensitizes leptin signal transduction and the subsequent downstream anorexic and thermogenic responses in normal and leptin-resistant age-related obese rats. To this end, we administered, i.c.v., recombinant adeno-associated virus encoding rat leptin cDNA (rAAV-leptin) or control virus into young and aged-obese rats and after 9 or 46 days, examined food intake, oxygen consumption, body weight, serum leptin, STAT3 phosphorylation, hypothalamic NPY and POMC mRNAs, and UCP1 expression and protein level in brown adipose tissue (BAT). In young rats, rAAV-leptin depleted body fat and both anorexic and thermogenic mechanisms contributed to this effect. Moreover, leptin signal transduction was not desensitized, and there were persistent physiological responses. Similarly, in the aged-obese rats, there was unabated leptin signal transduction, however, both the anorexic and thermogenic responses completely attenuated sometime after day 9. This attenuation, downstream of the leptin receptor, may be contributing to the leptin-resistance and age-related weight gain in these aged-obese rats. Finally, in young rats, although the initial responses to rAAV-leptin were dominated by anorexic responses, by 46 days, the predominant response was thermogenic rather than anorexic, suggesting that energy expenditure may be an important component of long-term weight maintenance.

[Application of endolymphatic therapy using catheterization of femoral lymphatic vessels in complex of treatment of oncological diseases]. / Primenenie éndolimfaticheskoi terapii putem kateterizatsii limfaticheskikh sosudov bedra v komplekse lecheniia onkologicheskikh zabolevanii.

Endolymphatic way of the medicinal preparations administration promotes their more effective influence on pathological focus. The experience of conduction of more then 15,000 of the lymphatic vessels catheterization manipulations was summarized, permitting to perform the treatment of patients successfully.