Administration of an AAV vector coding for a P2X7-blocking nanobody-based biologic ameliorates colitis in mice.

BACKGROUND: The pro-inflammatory ATP-gated P2X7 receptor is widely expressed by immune and non-immune cells. Nanobodies targeting P2X7, with potentiating or antagonistic effects, have been developed. Adeno-associated virus (AAV)-mediated gene transfer represents an efficient approach to achieve long-term in vivo expression of selected nanobody-based biologics. This approach (AAVnano) was used to validate the relevance of P2X7 as a target in dextran sodium sulfate (DSS)-induced colitis in mice. RESULTS: Mice received an intramuscular injection of AAV vectors coding for potentiating (14D5-dimHLE) or antagonistic (13A7-Fc) nanobody-based biologics targeting P2X7. Long-term modulation of P2X7 activity was evaluated ex vivo from blood samples. Colitis was induced with DSS in mice injected with AAV vectors coding for nanobody-based biologics. Severity of colitis, colon histopathology and expression of chemokines and cytokines were determined to evaluate the impact of P2X7 modulation. A single injection of an AAV vector coding for 13A7-Fc or 14D5-dimHLE efficiently modulated P2X7 function in vivo from day 15 up to day 120 post-injection in a dose-dependent manner. An AAV vector coding for 13A7-Fc significantly ameliorated DSS-induced colitis and significantly reduced immune cell infiltration and expression of chemokines and proinflammatory cytokines in colonic tissue. CONCLUSIONS: We have demonstrated the validity of AAVnano methodology to modulate P2X7 functions in vivo. Applying this methodological approach to a DSS-induced colitis model, we have shown that P2X7 blockade reduces inflammation and disease severity. Hence, this study confirms the importance of P2X7 as a pharmacological target and suggests the use of nanobody-based biologics as potential therapeutics in inflammatory bowel disease.

Canadian guidance for diagnosis and management of acute hepatic porphyrias.

Acute hepatic porphyrias (AHP) comprise four rare monogenic autosomal conditions. Each is linked to a deficiency of heme metabolizing enzymes. Common manifestations include severe abdominal pain, nausea, confusion, hyponatremia, hypertension, tachycardia, and neuropathy. Diagnosis is challenging due to a non-specific, variable presentation with symptoms mimicking other common conditions. Initial diagnosis of AHP can be made with a test for urinary porphobilinogen, δ-aminolevulinic acid and porphyrins using a single random (spot) sample. However, many patients have complications due to delays in diagnosis and management. A novel small interfering RNA-based agent, givosiran, has demonstrated efficacy in reducing acute attacks in a recent Phase III trial, leading to its approval for the management of AHP. Early diagnosis is crucial for the timely introduction of disease-modifying treatments that reduce impairments, enhance quality of life, and extend survival. In this guidance, we aim to improve awareness and outcomes of AHP by making recommendations about diagnosis, monitoring, and treatment in Canada.

Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.

Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

'To improve quality of life': Diverging enactments of a value in nephrology clinical practices.

Quality of life has become a central value in the provision of healthcare for patients with chronic conditions. This has engendered debates in critical medical sociology on the non-neutral effects that valuing health and illness, medical interventions, and health care delivery in terms of quality of life yields. Focusing on the case of nephrology, this paper presents qualitative data collected in Austria of two dialysis units in which nephrologists initiated projects aimed towards 'the improvement of patients' quality of life'. Whereas the first involved nurses supporting patients in the administration of peritoneal dialysis at home, the second implied the provision of treatment and care exclusively focused on a well-being 'in the here and now' to patients. By conceptualising physicians as actors within networks of relations and values enacted in practices, it analyses how in both dialysis units reference to quality of life enabled nephrologists to problematise the provision of standard haemodialysis treatment to multi-morbid, elderly patients, to develop a new treatment protocol, and to interest and enrol others in the provision of healthcare according this new protocol. Valuing medical interventions in terms of quality of life not only leads to a governmentalization of living and an economisation of health. It also allows physicians to articulate a socio-medico-ethical problem - the availability of life-prolonging technologies for a growing population of elderly, multi-morbid patients - and develop solutions locally. What the solutions consist in may fundamentally differ, however.

Targeting multiple myeloma with nanobody-based heavy chain antibodies, bispecific killer cell engagers, chimeric antigen receptors, and nanobody-displaying AAV vectors.

Nanobodies are well suited for constructing biologics due to their high solubility. We generated nanobodies directed against CD38, a tumor marker that is overexpressed by multiple myeloma and other hematological malignancies. We then used these CD38-specific nanobodies to construct heavy chain antibodies, bispecific killer cell engagers (BiKEs), chimeric antigen receptor (CAR)-NK cells, and nanobody-displaying AAV vectors. Here we review the utility of these nanobody-based constructs to specifically and effectively target CD38-expressing myeloma cells. The promising results of our preclinical studies warrant further clinical studies to evaluate the potential of these CD38-specific nanobody-based constructs for treatment of multiple myeloma.

Enhanced Transduction of P2X7-Expressing Cells with Recombinant rAAV Vectors.

Adeno-associated viruses (AAV) are useful vectors for transducing cells in vitro and in vivo. Targeting of specific cell subsets with AAV is limited by the broad tropism of AAV serotypes. Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Their small size and high solubility allow easy reformatting into fusion proteins. In this chapter we provide protocols for inserting a P2X7-specific nanobody into a surface loop of the VP1 capsid protein of AAV2. Such nanobody-displaying recombinant AAV allow 50- to 500-fold stronger transduction of P2X7-expressing cells than the parental AAV. We provide protocols for monitoring the transduction of P2X7-expressing cells by nanobody-displaying rAAV by flow cytometry and fluorescence microscopy.

Evaluation of nanobody-based biologics targeting purinergic checkpoints in tumor models in vivo.

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. ATP promotes tumor growth but also anti-tumor immune responses notably via the P2X7 receptor. ATP can also be catabolized by CD39 and CD73 ecto-enzymes into immunosuppressive adenosine. P2X7, CD39 and CD73 have attracted much interest in cancer as targets offering the potential to unleash anti-tumor immune responses. These membrane proteins represent novel purinergic checkpoints that can be targeted by small drugs or biologics. Here, we investigated nanobody-based biologics targeting mainly P2X7, but also CD73, alone or in combination therapies. Blocking P2X7 inhibited tumor growth and improved survival of mice in cancer models that express P2X7. P2X7-potentiation by a nanobody-based biologic was not effective alone to control tumor growth but enhanced tumor control and immune responses when used in combination with oxaliplatin chemotherapy. We also evaluated a bi-specific nanobody-based biologic that targets PD-L1 and CD73. This novel nanobody-based biologic exerted a potent anti-tumor effect, promoting tumor rejection and improving survival of mice in two tumor models. Hence, this study highlights the importance of purinergic checkpoints in tumor control and open new avenues for nanobody-based biologics that may be further exploited in the treatment of cancer.

The voices of family caregivers of seniors with chronic conditions: a window into their experience using a qualitative design.

BACKGROUND: Family caregivers are the backbone of the healthcare system. Over time, caregiving takes a tremendous toll on the caregiver. This is particularly true for caregivers who (1) provide >21 h of care/week, and/or (2) support those experiencing depression, cognitive decline, aggressive behaviours, and life-limiting conditions requiring complex care. Many caregivers face deteriorating physical and mental health, social isolation, family conflict, and job loss. Caregivers often have little energy or time to access resources and their experiences with the healthcare system, healthcare professionals and service agencies can either buoy them through challenging times, or contribute further to their distress. OBJECTIVE: This project aimed to hear the voices of family caregivers; their challenges, struggles, joys, and motivation for persevering through hardship, as well as their recommendations regarding education, resources, and supports that might enhance their resilience. METHODS: This community engagement research project utilized an ethnographic, qualitative approach involving three, 2-h focus groups that were analyzed using thematic analysis. FINDINGS: Caregivers identified barriers to resilience, including demands on their time, changing roles and responsibilities, challenges of learning about medical conditions, their own emotional responses, financial strains, changing family dynamics, and personal health. Caregivers also identified several facilitators to resilience, including motivations for caregiving, sense of purpose and validation, spirituality, emotional experiences, and coping strategies. CONCLUSION: Caregivers recommended that educational opportunities, including increasing health care providers education concerning dementias, increased access to resources, system navigators, financial supports, political advocacy, and a more responsive caregiver centered system would support family caregiving.