The Role of Recombinant AAV in Precise Genome Editing

The replication-defective, non-pathogenic, nearly ubiquitous single-stranded adeno-associated viruses (AAVs) have gained importance since their discovery about 50 years ago. Their unique life cycle and virus-cell interactions have led to the development of recombinant AAVs as ideal genetic medicine tools that have evolved into effective commercialized gene therapies. A distinctive property of AAVs is their ability to edit the genome precisely. In contrast to all current genome editing platforms, AAV exclusively utilizes the high-fidelity homologous recombination (HR) pathway and does not require exogenous nucleases for prior cleavage of genomic DNA. Together, this leads to a highly precise editing outcome that preserves genomic integrity without incorporation of indel mutations or viral sequences at the target site while also obviating the possibility of off-target genotoxicity. The stem cell-derived AAV (AAVHSCs) were found to mediate precise and efficient HR with high on-target accuracy and at high efficiencies. AAVHSC editing occurs efficiently in post-mitotic cells and tissues in vivo. Additionally, AAV also has the advantage of an intrinsic delivery mechanism. Thus, this distinctive genome editing platform holds tremendous promise for the correction of disease-associated mutations without adding to the mutational burden. This review will focus on the unique properties of direct AAV-mediated genome editing and their potential mechanisms of action.

Scientific and Regulatory Policy Committee Points to Consider: Nonclinical Research and Development of In Vivo Gene Therapy Products, Emphasizing Adeno-Associated Virus Vectors

Sequencing of the human genome and numerous advances in molecular techniques have launched the era of genetic medicine. Increasingly precise technologies for genetic modification, manufacturing, and administration of pharmaceutical-grade biologics have proved the viability of in vivo gene therapy (GTx) as a therapeutic modality as shown in several thousand clinical trials and recent approval of several GTx products for treating rare diseases and cancers. In recognition of the rapidly advancing knowledge in this field, the regulatory landscape has evolved considerably to maintain appropriate monitoring of safety concerns associated with this modality. Nonetheless, GTx safety assessment remains complex and is designed on a case-by-case basis that is determined by the disease indication and product attributes. This article describes our current understanding of fundamental biological principles and possible procedures (emphasizing those related to toxicology and toxicologic pathology) needed to support research and development of in vivo GTx products. This article is not intended to provide comprehensive guidance on all GTx modalities but instead provides an overview relevant to in vivo GTx generally by utilizing recombinant adeno-associated virus-based GTx—the most common in vivo GTx platform—to exemplify the main points to be considered in nonclinical research and development of GTx products.

How is family health history discussed in routine primary healthcare? A qualitative study of archived family doctor consultations

ObjectivesFamily health history underpins genetic medicine. Our study aimed to explore language and patterns of communication relating to family health history observed in interactions between general practitioners (GPs) and their patients within routine primary care consultations.DesignSecondary analysis of patient and GP routine consultation data (n=252).ParticipantsConsultations that included ‘family health history’ were eligible for inclusion (n=58).Primary outcomesA qualitative inductive analysis of the interactions from consultation transcripts.Results46/58 conversations about family health history were initiated by the GP. Most discussions around family history lasted for between approximately 1 to 2 min. Patients were invited to share family health history through one of two ways: non-specific enquiry (eg, by asking the patient about ‘anything that runs in the family’); or specific enquiry where they were asked if they had a ‘strong family history’ in relation to a particular condition, for example, breast cancer. Patients often responded to either approach with a simple no, but fuller negative responses also occurred regularly and typically included an account of some kind (eg, explaining family relationships/dynamics which impeded or prevented the accessibility of information).ConclusionsFamily health history is regarded as a genetic test and is embedded in the sociocultural norms of the patient from whom information is being sought. Our findings highlight that it is more complex than asking simply if ‘anything’ runs in the family. As the collection of family health history is expected to be more routine, it will be important to also consider it from sociocultural perspectives in order to help mitigate any inequities in how family history is collected, and therefore used (or not) in a person’s healthcare. Orientating an enquiry away from ‘anything’ and asking more specific details about particular conditions may help facilitate the dialogue.

Improving turnaround times for HLA-B*27 and HLA-B*57:01 gene testing: a Barts Health NHS Trust quality improvement project

Among other tests, Barts Health NHS Trust clinical transplantation laboratory conducts two important gene-detection tests: human leucocyte antigen (HLA)-B*27 (‘B27’, associated with the diagnosis of ankylosing spondylitis) and HLA-B*57:01 (‘B57’, associated with prediction of abacavir hypersensitivity disorder). The turnaround time (TaT) from sample receipt to return of results is important to clinicians and their patients but was not monitored. Furthermore, we anticipated an imminent increase in demand from a forthcoming pathology service merger, together with long-term increases with the rise of personalised genetic medicine.In this quality improvement project, we identified current TaT performance and sources of delay. Over three plan-do-study-act (PDSA) cycles, we tested three change ideas, two involving using IT to remove manual administrative steps and alert us to samples needing progressing; both were retained. The other change involved separating out the targeted tests; we judged this not worthwhile with current demand levels, although something to be re-examined when volumes increase. During the project, we reduced mean TaT from 3.8 to 3.3 days and increased the proportion within our 5-day target from 78% to 100%. These have been sustained (at 3.4 days and 97%) for the 3 months following our PDSA cycles and illustrate that reducing variation can be as impactful as reducing the mean.We conducted this project during the COVID-19 disruption, which reduced demand substantially. We took advantage of this to allow staff to spend time on these improvement activities. Another interesting feature of the work is that during the project, we compared changes in performance on our targeted B27/B57 tests with that on another comparable test as a control, to consider the impact of the general increased attention (the Hawthorne effect). We found that performance on this control also increased comparably, but then fell away after our project finished, while it did not for B27/B57.

CoverageMaster: comprehensive CNV detection and visualization from NGS short reads for genetic medicine applications

CoverageMaster (CoM) is a Copy Number Variation (CNV) calling algorithm based on depth-of-coverage maps designed to detect CNVs of any size in exome (WES) and genome (WGS) data. The core of the algorithm is the compression of sequencing coverage data in a multiscale Wavelet space and the analysis through an iterative Hidden Markov Model (HMM). CoM processes WES and WGS data at nucleotide scale resolution and accurately detect and visualize full size range CNVs, including single or partial exon deletions and duplications. The results obtained with this approach support the possibility for coverage-based CNV callers to replace probe-based methods such array CGH and MLPA in the near future.

Electronic Medical Record and Clinical Evaluation of Two Patient Cohorts at Risk of Hypophosphatasia: Family History is an Underutilized Tool to Diagnose Patients with Rare Disease (Preprint)

BACKGROUND Family history (FH) is a powerful tool in screening and testing for chronic disease, oncologic conditions, and other genetic diagnoses, and is dependent upon observing patterns of features inherited across generations. Despite acceptance of FH as a fundamental part of a genetic medicine evaluation, there are limited data in other medical fields about its utility and benefit to patients. To date, there are no published data pertaining to the clinical utility of FH to diagnose individuals with hypophosphatasia (HPP), a rare genetic low bone density condition. OBJECTIVE To compare FH recorded in the electronic medical record (EMR) of patients at risk of HPP before a genetic medicine encounter to that learned during that clinical evaluation. METHODS We evaluated two patient populations at-risk of a rare metabolic condition-- hypophosphatasia (HPP)—for the quality and quantity of FH data in their medical records. Population 1 was derived from patients seeking evaluation for a low bone density diagnosis at the Johns Hopkins Greenberg Center for Skeletal Dysplasias (GCSD); population 2 was identified through a targeted electronic medical record (EMR) query of low serum alkaline phosphatase (AP) measurements obtained through the Johns Hopkins Clinical Laboratory. RESULTS In population 1 (n = 38), 27 (71%) were confirmed to have HPP. Of these, 14 (52%) presented with FH information in the EMR from 3 or more family members, and in 4 (15%) FH was suggestive of HPP. In population 2 (n=348), a similar proportion of subjects had 3 or more family members mentioned in the EMR (183 or 53%), but only 3 subjects (1%) included sufficient detail to determine that the family history was suggestive of HPP. Notably, once all patients in population 1 completed a medical genetics evaluation and HPP was confirmed, 20 (74%) had probable affected family members identified through obtaining and analyzing a pedigree. After cascade testing was offered to these family members, 17 patients (71%) from population 1 had at least one family member with HPP confirmed at a molecular level. CONCLUSIONS Based on these results, we propose that a full genetic medicine evaluation of those subjects from population 2 at highest risk of HPP would confirm HPP in many of them and identify similarly affected family members. We propose that taking a FH refines the diagnostic precision for patients with low bone density and identifies affected family members, and that HPP is likely more prevalent than previously thought. CLINICALTRIAL n/a