Quantitative assessment of barriers to the clinical development and adoption of cellular therapies:A pilot study

There has been a large increase in basic science activity in cell therapy and a growing portfolio of cell therapy trials. However, the number of industry products available for widespread clinical use does not match this magnitude of activity. We hypothesize that the paucity of engagement with the clinical community is a key contributor to the lack of commercially successful cell therapy products. To investigate this, we launched a pilot study to survey clinicians from five specialities and to determine what they believe to be the most significant barriers to cellular therapy clinical development and adoption. Our study shows that the main concerns among this group are cost-effectiveness, efficacy, reimbursement, and regulation. Addressing these concerns can best be achieved by ensuring that future clinical trials are conducted to adequately answer the questions of both regulators and the broader clinical community

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome.METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants.RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving.CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.</p

Single-dose BNT162b2 vaccine protects against asymptomatic SARS-CoV-2 infection

The BNT162b2 mRNA COVID-19 vaccine (Pfizer-BioNTech) is being utilised internationally for mass COVID-19 vaccination. Evidence of single-dose protection against symptomatic disease has encouraged some countries to opt for delayed booster doses of BNT162b2, but the effect of this strategy on rates of asymptomatic SARS-CoV-2 infection remains unknown. We previously demonstrated frequent pauci- and asymptomatic SARS-CoV-2 infection amongst healthcare workers (HCWs) during the UK’s first wave of the COVID-19 pandemic, using a comprehensive PCR-based HCW screening programme (Rivett et al., 2020; Jones et al., 2020). Here, we evaluate the effect of first-dose BNT162b2 vaccination on test positivity rates and find a fourfold reduction in asymptomatic infection amongst HCWs ≥12 days post-vaccination. These data provide real-world evidence of short-term protection against asymptomatic SARS-CoV-2 infection following a single dose of BNT162b2 vaccine, suggesting that mass first-dose vaccination will reduce SARS-CoV-2 transmission, as well as the burden of COVID-19 disease

Identification of a Cryptic Bacterial Promoter in Mouse (<i>mdr1a</i>) P-Glycoprotein cDNA

<div><p>The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse <i>mdr1a</i> cDNA display significant genetic instability during cloning in bacteria, indicating that <i>mdr1a</i> cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse <i>mdr1a</i> cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of <i>mdr1a</i> DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of <i>mdr1a</i> cDNA capable of initiating bacterial protein expression. An <i>mdr1a</i> M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i> while retaining transport properties similar to wild-type P-gp. This mutant <i>mdr1a</i> cDNA may prove useful for efficient cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i>.</p></div

Persistence and efficacy of second generation CAR T Cell against the LeY Antigen in acute myeloid leukemia

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence

Identification of putative transcriptional and translational start sites in <i>mdr1a</i> cDNA.

<p>An <i>in silico</i> sigma 70 binding site analysis was used to screen for a putative cryptic bacterial promoter in <i>mdr1a</i> cDNA. (A) Sequence of <i>mdr1a</i> cDNA shown with predicted -35 and extended -10 elements (red). The location in the cDNA for each element is noted above the sequence. (B) Predicted Shine-Dalgarno and ATG of a methionine that is in-frame with regards to the <i>mdr1a</i> ORF.</p

Mutations and their position in <i>mdr1a</i> cDNA after transformation into <i>E</i>. <i>coli</i>.

<p>The pCR-Blunt-II-TOPO vector containing <i>mdr1a</i> cDNA was used to transform <i>E</i>.<i>coli</i> and 20 colonies were selected for small-scale bacterial growth, plasmid purification, and analysis of plasmid DNA by agarose gel electrophoresis. Sixteen of the colonies selected for analysis contained large insertion or deletion mutations (not shown), and the remaining four colonies containing potentially correct <i>mdr1a</i> cDNA were sequenced. Each of the four sequenced plasmids contained mutated <i>mdr1a</i> cDNA, indicating a 100% mutational rate. Mutations observed were classified as point, insertion, or deletion mutations. Wild-type <i>mdr1a</i> cDNA (top) is compared to sequenced, “mutant” <i>mdr1a</i> cDNA (bottom). Insertion and point mutations are indicated in red in the mutant cDNA. Deleted base pairs are highlighted in red in wild-type cDNA, and the resulting mutated cDNA sequence is shown below. A) A point mutation (C→T) at location 1027 bp resulting in an introduced stop codon into <i>mdr1a</i> cDNA. B) A single adenine base pair insertion at location 1033 bp. C) Two examples of deletion mutations. All mutations resulted either directly (point mutations) or indirectly (insertion or deletion mutations) in the introduction of a stop codon into <i>mdr1a</i> cDNA. Locations of the introduced stop codons are indicated.</p