Highlights d A SARS-CoV-2 variant with Spike G614 has replaced D614 as the dominant pandemic form d The consistent increase of G614 at regional levels may indicate a fitness advantage d G614 is associated with lower RT PCR Cts, suggestive of higher viral loads in patients d The G614 variant grows to higher titers as pseudotyped virions

Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

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Spinal muscular atrophy is one of the most common genetic causes of death in childhood, and there is currently no effective treatment. The disease is caused by mutations in the survival motor neuron gene. Gene therapy aimed at restoring the protein encoded by this gene is a rational therapeutic approach to ameliorate the disease phenotype. We previously reported that intramuscular delivery of a lentiviral vector expressing survival motor neuron increased the life expectancy of transgenic mice with spinal muscular atrophy. The marginal efficacy of this therapeutic approach, however, prompted us to explore different strategies for gene therapy delivery to motor neurons to achieve a more clinically relevant effect. Here, we report that a single injection of self-complementary adeno-associated virus serotype 9 expressing green fluorescent protein or of a codon-optimized version of the survival motor neuron protein into the facial vein 1 day after birth in mice carrying a defective survival motor neuron gene led to widespread gene transfer. Furthermore, this gene therapy resulted in a substantial extension of life span in these animals. These data demonstrate a significant increase in survival in a mouse model of spinal muscular atrophy and provide evidence for effective therapy.

ObjectiveAn intronic GGGGCC-repeat expansion of C9ORF72 is the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The mechanism of neurodegeneration is unknown, but a direct effect on RNA processing mediated by RNA foci transcribed from the repeat sequence has been proposed.MethodsGene expression profiling utilised total RNA extracted from motor neurons and lymphoblastoid cell lines derived from human ALS patients, including those with an expansion of C9ORF72, and controls. In lymphoblastoid cell lines, expansion length and the frequency of sense and antisense RNA foci was also examined.ResultsGene level analysis revealed a number of differentially expressed networks and both cell types exhibited dysregulation of a network functionally enriched for genes encoding ‘RNA splicing’ proteins. There was a significant overlap of these genes with an independently generated list of GGGGCC-repeat protein binding partners. At the exon level, in lymphoblastoid cells derived from C9ORF72-ALS patients splicing consistency was lower than in lines derived from non-C9ORF72 ALS patients or controls; furthermore splicing consistency was lower in samples derived from patients with faster disease progression. Frequency of sense RNA foci showed a trend towards being higher in lymphoblastoid cells derived from patients with shorter survival, but there was no detectable correlation between disease severity and DNA expansion length.SignificanceUp-regulation of genes encoding predicted binding partners of the C9ORF72 expansion is consistent with an attempted compensation for sequestration of these proteins. A number of studies have analysed changes in the transcriptome caused by C9ORF72 expansion, but to date findings have been inconsistent. As a potential explanation we suggest that dynamic sequestration of RNA processing proteins by RNA foci might lead to a loss of splicing consistency; indeed in our samples measurement of splicing consistency correlates with disease severity.