Life-Limiting Peripheral Organ Dysfunction in Feline Sandhoff Disease Emerges after Effective CNS Gene Therapy.

OBJECTIVE: GM2 gangliosidosis is usually fatal by 5 years of age in its 2 major subtypes, Tay-Sachs and Sandhoff disease. First reported in 1881, GM2 gangliosidosis has no effective treatment today, and children succumb to the disease after a protracted neurodegenerative course and semi-vegetative state. This study seeks to further develop adeno-associated virus (AAV) gene therapy for human translation. METHODS: Cats with Sandhoff disease were treated by intracranial injection of vectors expressing feline ß-N-acetylhexosaminidase, the enzyme deficient in GM2 gangliosidosis. RESULTS: Hexosaminidase activity throughout the brain and spinal cord was above normal after treatment, with highest activities at the injection sites (thalamus and deep cerebellar nuclei). Ganglioside storage was reduced throughout the brain and spinal cord, with near complete clearance in many regions. While untreated cats with Sandhoff disease lived for 4.4 ± 0.6 months, AAV-treated cats lived to 19.1 ± 8.6 months, and 3 of 9 cats lived >21 months. Correction of the central nervous system was so effective that significant increases in lifespan led to the emergence of otherwise subclinical peripheral disease, including megacolon, enlarged stomach and urinary bladder, soft tissue spinal cord compression, and patellar luxation. Throughout the gastrointestinal tract, neurons of the myenteric and submucosal plexuses developed profound pathology, demonstrating that the enteric nervous system was inadequately treated. INTERPRETATION: The vector formulation in the current study effectively treats neuropathology in feline Sandhoff disease, but whole-body targeting will be an important consideration in next-generation approaches. ANN NEUROL 2023;94:969-986.

Discharging to the Street: When Patients Refuse Medically Safer Options.

The ethical obligation to provide a reasonably safe discharge option from the inpatient setting is often confounded by the context of homelessness. Living without the security of stable housing is a known determinant of poor health, often complicating the safety of discharge and causing unnecessary readmission. But clinicians do not have significant control over unjust distributions of resources or inadequate societal investment in social services. While physicians may stretch inpatient stays beyond acute care need in the interest of their patients who are experiencing homelessness, they must also consider the implications of using an inpatient hospital bed for someone without the attendant level of medical need. Caring for patients in an inpatient setting when they no longer require acute care means fewer beds for acute care patients. And when a patient who is experiencing homelessness declines a medically safer option such as a skilled nursing facility, then clinicians may be faced with the sole option of discharge to the street, which raises troubling questions of nonmaleficence and social justice. Here we investigate the different forms of injustice that play out when patients are discharged to the street, and offer a map of the interwoven ethical responsibilities of clinicians, hospitals, and skilled nursing facilities.

Immune Responses to Viral Gene Therapy Vectors.

Several viral vector-based gene therapy drugs have now received marketing approval. A much larger number of additional viral vectors are in various stages of clinical trials for the treatment of genetic and acquired diseases, with many more in pre-clinical testing. Efficiency of gene transfer and ability to provide long-term therapy make these vector systems very attractive. In fact, viral vector gene therapy has been able to treat or even cure diseases for which there had been no or only suboptimal treatments. However, innate and adaptive immune responses to these vectors and their transgene products constitute substantial hurdles to clinical development and wider use in patients. This review provides an overview of the type of immune responses that have been documented in animal models and in humans who received gene transfer with one of three widely tested vector systems, namely adenoviral, lentiviral, or adeno-associated viral vectors. Particular emphasis is given to mechanisms leading to immune responses, efforts to reduce vector immunogenicity, and potential solutions to the problems. At the same time, we point out gaps in our knowledge that should to be filled and problems that need to be addressed going forward.

Type I IFN Sensing by cDCs and CD4+ T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8+ T Cells.

Adeno-associated virus (AAV) vectors are widely used in clinical gene therapy to correct genetic disease by in vivo gene transfer. Although the vectors are useful, in part because of their limited immunogenicity, immune responses directed at vector components have complicated applications in humans. These include, for instance, innate immune sensing of vector components by plasmacytoid dendritic cells (pDCs), which sense the vector DNA genome via Toll-like receptor 9. Adaptive immune responses employ antigen presentation by conventional dendritic cells (cDCs), which leads to cross-priming of capsid-specific CD8+ T cells. In this study, we sought to determine the mechanisms that promote licensing of cDCs, which is requisite for CD8+ T cell activation. Blockage of type 1 interferon (T1 IFN) signaling by monoclonal antibody therapy prevented cross-priming. Furthermore, experiments in cell-type-restricted knockout mice showed a specific requirement for the receptor for T1 IFN (IFNaR) in cDCs. In contrast, natural killer (NK) cells are not needed, indicating a direct rather than indirect effect of T1 IFN on cDCs. In addition, co-stimulation by CD4+ T cells via CD40-CD40L was required for cross-priming, and blockage of co-stimulation but not of T1 IFN additionally reduced antibody formation against capsid. These mechanistic insights inform the development of targeted immune interventions.

Making Medical Treatment Decisions for Unrepresented Patients in the ICU. An Official American Thoracic Society/American Geriatrics Society Policy Statement.

Background and Rationale: ICU clinicians regularly care for patients who lack capacity, an applicable advance directive, and an available surrogate decision-maker. Although there is no consensus on terminology, we refer to these patients as "unrepresented." There is considerable controversy about how to make treatment decisions for these patients, and there is significant variability in both law and clinical practice.Purpose and Objectives: This multisociety statement provides clinicians and hospital administrators with recommendations for decision-making on behalf of unrepresented patients in the critical care setting.Methods: An interprofessional, multidisciplinary expert committee developed this policy statement by using an iterative consensus process with a diverse working group representing critical care medicine, palliative care, pediatric medicine, nursing, social work, gerontology, geriatrics, patient advocacy, bioethics, philosophy, elder law, and health law.Main Results: The committee designed its policy recommendations to promote five ethical goals: 1) to protect highly vulnerable patients, 2) to demonstrate respect for persons, 3) to provide appropriate medical care, 4) to safeguard against unacceptable discrimination, and 5) to avoid undue influence of competing obligations and conflicting interests. These recommendations also are intended to strike an appropriate balance between excessive and insufficient procedural safeguards. The committee makes the following recommendations: 1) institutions should offer advance care planning to prevent patients at high risk for becoming unrepresented from meeting this definition; 2) institutions should implement strategies to determine whether seemingly unrepresented patients are actually unrepresented, including careful capacity assessments and diligent searches for potential surrogates; 3) institutions should manage decision-making for unrepresented patients using input from a diverse interprofessional, multidisciplinary committee rather than ad hoc by treating clinicians; 4) institutions should use all available information on the patient's preferences and values to guide treatment decisions; 5) institutions should manage decision-making for unrepresented patients using a fair process that comports with procedural due process; 6) institutions should employ this fair process even when state law authorizes procedures with less oversight.Conclusions: This multisociety statement provides guidance for clinicians and hospital administrators on medical decision-making for unrepresented patients in the critical care setting.

Plasmacytoid and conventional dendritic cells cooperate in crosspriming AAV capsid-specific CD8+ T cells.

Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8+ T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8+ T cells against the input viral capsid antigen. In this study, we report that the Toll-like receptor 9 (TLR9)-MyD88 pattern-recognition receptor pathway is uniquely capable of initiating this response. By contrast, the absence of TLR2, STING, or the addition of TLR4 agonist has no effect. Surprisingly, both conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are required for the crosspriming of capsid-specific CD8+ T cells, whereas other antigen-presenting cells are not involved. TLR9 signaling is specifically essential in pDCs but not in cDCs, indicating that sensing of the viral genome by pDCs activates cDCs in trans to cross-present capsid antigen during CD8+ T-cell activation. Cross-presentation and crosspriming depend not only on TLR9, but also on interferon type I signaling, and both mechanisms can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8+ T cells. Thus, these outcomes directly point to therapeutic interventions and demonstrate that innate immune blockade can eliminate unwanted immune responses in gene therapy.

Novel Biomarkers of Human GM1 Gangliosidosis Reflect the Clinical Efficacy of Gene Therapy in a Feline Model.

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.

No Need to Object: Ethical Obligations for Interprofessional Collaboration in Emergency Department Discharge Planning.

Emergency departments (EDs) serve a wide range of patient needs. A crucial aspect of safe and effective care in the ED is to appropriately transition patients to the next level of care. In most EDs, this disposition planning is done exclusively by physicians, which has the potential to result in unacceptable harm. A virtue ethics approach demonstrates the need for explicit inclusion of nurses in disposition planning. In utilizing this approach, it is necessary to examine four focal virtues as they relate to the work of disposition planning and the moral character of the nurse. The virtues of prudence, trustworthiness, vigilance, and courage show that interprofessional collaboration is needed during disposition planning to promote patient safety, facilitate interprofessional relationships, and prevent moral distress. The majority of literature on disposition planning is empirical in nature; this chapter adds a normative argument and a motive for policy reform.

Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy.

Adeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector's DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.

TLR9-Activating CpG-B ODN but Not TLR7 Agonists Triggers Antibody Formation to Factor IX in Muscle Gene Transfer.

Innate immune signals that promote B cell responses in gene transfer are generally ill-defined. In this study, we evaluate the effect of activating endosomal Toll-like receptors 7, 8, and 9 (TLR7, TLR7/8, and TLR9) on antibody formation during muscle-directed gene therapy with adeno-associated virus (AAV) vectors. We examined whether activation of endosomal TLRs, by adenine analog CL264 (TLR7 agonist), imidazolquinolone compound R848 (TLR7/8 agonist), or class B CpG oligodeoxynucleotides ODN1826 (TLR9 agonist), could augment antibody formation upon intramuscular administration of AAV1 expressing human clotting factor IX (AAV1-hFIX) in mice. The TLR9 agonist robustly enhanced antibody formation by the 1st week, thus initially eliminating systemic hFIX expression. By contrast, the TLR7 and TLR7/8 agonists did not markedly promote antibody formation, or significantly reduce circulating hFIX. We concurrently investigated the effects of these TLR agonists during muscle gene transfer on mature B cells and dendritic cells (DCs) in the draining lymph nodes including conventional DCs (CD11b+ or CD8α+ cDCs), monocyte-derived dendritic cells (moDCs), and plasmacytoid dendritic cells (pDCs). Only TLR9 stimulation caused a striking increase in the frequency of moDCs within 24 h. The TLR7/8 and TLR9 agonists activated pDCs, both subsets of cDCs, and mature B cells, whereas the TLR7 agonist had only mild effects on these cells. Thus, these TLR ligands have distinct effects on DCs and mature B cells, yet only the TLR9 agonist enhanced the humoral immune response against AAV-expressed hFIX. These new findings indicate a unique ability of certain TLR9 agonists to stimulate B cell responses in muscle gene transfer through enrichment of moDCs.

Biomarkers for disease progression and AAV therapeutic efficacy in feline Sandhoff disease.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme ß-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were largely normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated SD patients and those in future clinical trials.

Limits of autonomy in nursing's moral discourse.

This article critiques the dominance of the discourse of autonomy in nursing ethical conversations, and the ways in which it is linked with nursing's commitment to patient advocacy. In its current discursive form, autonomy is too often used to reinforce the position of those already privileged and exacerbates the challenges faced by those with social vulnerabilities. Nursing's historical identity provides both challenges and resources for developing alternate ethical values. We have allowed narrow understandings of autonomy and advocacy to dominate our ethical discourse and have relied too exclusively on them to resolve conflicts and guide practice.

Enhancing women's mood and energy: a research-based program for subthreshold depression using light, exercise, and vitamins.

The prevalence and clinical significance of subthreshold forms of depression with sequelae comparable to major depression have been recently described in the literature; however, research on effective treatment is rare. A new intervention program that combines a specific regimen of light, exercise, and vitamins is effective in improving women's mood and overall sense of well-being. This program is well suited to many patients who present with somatic and psychological symptoms consistent with subthreshold depression.