Suppression of matrigel-induced choroidal neovascularization by AAV delivery of a novel anti-Scg3 antibody
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Evolving adeno-associated viruses for gene transfer to the kidney via cross-species cycling of capsid libraries
The difficulty of delivering genes to the kidney has limited the translation of genetic medicines, particularly for the more than 10% of the global population with chronic kidney disease. Here we show that new variants of adeno-associated viruses (AAVs) displaying robust and widespread transduction in the kidneys of mice, pigs and non-human-primates can be obtained by evolving capsid libraries via cross-species cycling in different kidney models. Specifically, the new variants, AAV.k13 and AAV.k20, were enriched from the libraries following sequential intravenous cycling through mouse and pig kidneys, ex vivo cycling in human organoid cultures, and ex vivo machine perfusion in isolated kidneys from rhesus macaques. The two variants transduced murine kidneys following intravenous administration, with selective tropism for proximal tubules, and led to markedly higher transgene expression than parental AAV9 vectors in proximal tubule epithelial cells within human organoid cultures and in autotransplanted pig kidneys. Following ureteral delivery, AAV.k20 efficiently transduced kidneys in pigs and macaques. The AAV.k13 and AAV.k20 variants are promising vectors for therapeutic gene-transfer applications in kidney diseases and transplantation.
AAV capsid prioritization in normal and steatotic human livers maintained by machine perfusion
Therapeutic efficacy and safety of adeno-associated virus (AAV) liver gene therapy depend on capsid choice. To predict AAV capsid performance under near-clinical conditions, we established side-by-side comparison at single-cell resolution in human livers maintained by normothermic machine perfusion. AAV-LK03 transduced hepatocytes much more efficiently and specifically than AAV5, AAV8 and AAV6, which are most commonly used clinically, and AAV-NP59, which is better at transducing human hepatocytes engrafted in immune-deficient mice. AAV-LK03 preferentially transduced periportal hepatocytes in normal liver, whereas AAV5 targeted pericentral hepatocytes in steatotic liver. AAV5 and AAV8 transduced liver sinusoidal endothelial cells as efficiently as hepatocytes. AAV capsid and steatosis influenced vector episome formation, which determines gene therapy durability, with AAV5 delaying concatemerization. Our findings inform capsid choice in clinical AAV liver gene therapy, including consideration of disease-relevant hepatocyte zonation and effects of steatosis, and facilitate the development of AAV capsids that transduce hepatocytes or other therapeutically relevant cell types in the human liver with maximum efficiency and specificity.
Raptin, a sleep-induced hypothalamic hormone, suppresses appetite and obesity
Sleep deficiency is associated with obesity, but the mechanisms underlying this connection remain unclear. Here, we identify a sleep-inducible hypothalamic protein hormone in humans and mice that suppresses obesity. This hormone is cleaved from reticulocalbin-2 (RCN2), and we name it Raptin. Raptin release is timed by the circuit from vasopressin-expressing neurons in the suprachiasmatic nucleus to RCN2-positive neurons in the paraventricular nucleus. Raptin levels peak during sleep, which is blunted by sleep deficiency. Raptin binds to glutamate metabotropic receptor 3 (GRM3) in neurons of the hypothalamus and stomach to inhibit appetite and gastric emptying, respectively. Raptin-GRM3 signaling mediates anorexigenic effects via PI3K-AKT signaling. Of note, we verify the connections between deficiencies in the sleeping state, impaired Raptin release, and obesity in patients with sleep deficiency. Moreover, humans carrying an RCN2 nonsense variant present with night eating syndrome and obesity. These data define a unique hormone that suppresses food intake and prevents obesity.
Astrocytic cannabinoid receptor 1 promotes resilience by dampening stress-induced blood–brain barrier alterations
Blood–brain barrier (BBB) alterations contribute to stress vulnerability and the development of depressive behaviors. In contrast, neurovascular adaptations underlying stress resilience remain unclear. Here we report that high expression of astrocytic cannabinoid receptor 1 (CB1) in the nucleus accumbens (NAc) shell, particularly in the end-feet ensheathing blood vessels, is associated with resilience during chronic social stress in adult male mice. Viral-mediated overexpression of Cnr1 in astrocytes of the NAc shell results in baseline anxiolytic effects and dampens stress-induced anxiety- and depression-like behaviors in male mice. It promotes the expression of vascular-related genes and reduces astrocyte inflammatory response and morphological changes following an immune challenge with the cytokine interleukin-6, linked to stress susceptibility and mood disorders. Physical exercise and antidepressant treatment increase the expression of astrocytic Cnr1 in the perivascular region in male mice. In human tissue from male donors with major depressive disorder, we observe loss of CNR1 in the NAc astrocytes. Our findings suggest a role for the astrocytic endocannabinoid system in stress responses via modulation of the BBB.
Adeno-associated virus serotype 2 capsids with proteolytic cuts by trypsin remain intact and potent
Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. In the journey of an AAV vector, AAV vectors can be exposed to different proteolytic environments inside the production cells, during the cell lysis step, within the endosome, and finally inside the cell nucleus. The stability of a modified AAV serotype 2 (AAV2) capsid was evaluated via a proteolytic approach using trypsin and other proteases and both denaturing and non-denaturing analytical methods. Trypsin digestion of the AAV2 capsids resulted in clips of the capsid proteins at the C-terminus as confirmed by denaturing methods including SDS-PAGE, CE-SDS, Western blot, and RPLC-MS. It was found that the AAV2 capsid with clips not only remains structurally intact, as confirmed by non-denaturing methods including SEC, thermostability testing, and cryo-EM, but also remains potent, as confirmed in a cell-based potency assay. This finding reveals that AAV2 capsid with proteolytic cuts remains intact and potent since the icosahedral three-dimensional structural arrangement of AAV capsid proteins can protect the clipped fragment from being released from the capsid, such that the AAV capsid remains intact allowing for the functionality to be maintained to deliver the DNA in the host cell. Evaluation of AAV stability using a proteolytic approach and multiple denaturing and non-denaturing analytical methods can provide valuable information for engineering AAV capsids to develop AAV-based gene therapy.
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