Relaxing the restricted structural dynamics in the human hepatitis B virus RNA encapsidation signal enables replication initiation in vitro.

Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P-ε binding but no detectable priming. Here we demonstrate that local relaxation of the highly basepaired ε upper stem, by mutation or via synthetic split RNAs, enables ε-dependent in vitro priming with full-length P protein from eukaryotic translation extract yet also, and without additional macromolecules, with truncated HBV miniP proteins expressed in bacteria. Using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) we confirm that upper stem destabilization correlates with in vitro priming competence and show that the supposed bulge-closing basepairs are largely unpaired even in wild-type ε. We define the two 3´ proximal nt of this extended bulge as main initiation sites and provide evidence for a Dε-like opening of the apical ε part upon P protein binding. Beyond new HBV-specific basic aspects our novel in vitro priming systems should facilitate the development of high-throughput screens for priming inhibitors targeting this highly virus-specific process.

Effect of nusinersen on motor, respiratory and bulbar function in early-onset spinal muscular atrophy.

5q-associated spinal muscular atrophy is a rare neuromuscular disorder with the leading symptom of a proximal muscle weakness. Three different drugs have been approved by the European Medicines Agency and Food and Drug Administration for the treatment of spinal muscular atrophy patients, however, long-term experience is still scarce. In contrast to clinical trial data with restricted patient populations and short observation periods, we report here real-world evidence on a broad spectrum of patients with early-onset spinal muscular atrophy treated with nusinersen focusing on effects regarding motor milestones, and respiratory and bulbar insufficiency during the first years of treatment. Within the SMArtCARE registry, all patients under treatment with nusinersen who never had the ability to sit independently before the start of treatment were identified for data analysis. The primary outcome of this analysis was the change in motor function evaluated with the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders and motor milestones considering World Health Organization criteria. Further, we evaluated data on the need for ventilator support and tube feeding, and mortality. In total, 143 patients with early-onset spinal muscular atrophy were included in the data analysis with a follow-up period of up to 38 months. We observed major improvements in motor function evaluated with the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders. Improvements were greater in children >2 years of age at start of treatment than in older children. 24.5% of children gained the ability to sit independently. Major improvements were observed during the first 14 months of treatment. The need for intermittent ventilator support and tube feeding increased despite treatment with nusinersen. Our findings confirm the increasing real-world evidence that treatment with nusinersen has a dramatic influence on disease progression and survival in patients with early-onset spinal muscular atrophy. Major improvements in motor function are seen in children younger than 2 years at the start of treatment. Bulbar and respiratory function needs to be closely monitored, as these functions do not improve equivalent to motor function.

Improvements in Walking Distance during Nusinersen Treatment - A Prospective 3-year SMArtCARE Registry Study.

BACKGROUND AND OBJECTIVES: Disease progression in patients with spinal muscular atrophy (SMA) has changed dramatically within the past years due to the approval of three different disease-modifying treatments. Nusinersen was the first drug to be approved for the treatment of SMA patients. Clinical trials provided data from infants with SMA type 1 and children with SMA type 2, but there is still insufficient evidence and only scarcely reported long-term experience for nusinersen treatment in ambulant patients. Here, we report data from the SMArtCARE registry of ambulant patients under nusinersen treatment with a follow-up period of up to 38 months. METHODS: SMArtCARE is a disease-specific registry in Germany, Austria and Switzerland. Data are collected as real-world data during routine patient visits. Our analysis included all patients under treatment with nusinersen able to walk independently before start of treatment with focus on changes in motor function. RESULTS: Data from 231 ambulant patients were included in the analysis. During the observation period, 31 pediatric walkers (27.2%) and 31 adult walkers (26.5%) experienced a clinically meaningful improvement of≥30 m in the 6-Minute-Walk-Test. In contrast, only five adult walkers (7.7%) showed a decline in walking distance≥30 m, and two pediatric walkers (1.8%) lost the ability to walk unassisted under treatment with nusinersen. HFMSE and RULM scores improved in pediatric and remained stable in adult patients. CONCLUSION: Our data demonstrate a positive effect of nusinersen treatment in most ambulant pediatric and adult SMA patients. We not only observed a stabilization of disease progression or lack of deterioration, but clinically meaningful improvements in walking distance.

Improved upper limb function in non-ambulant children with SMA type 2 and 3 during nusinersen treatment: a prospective 3-years SMArtCARE registry study.

BACKGROUND: The development and approval of disease modifying treatments have dramatically changed disease progression in patients with spinal muscular atrophy (SMA). Nusinersen was approved in Europe in 2017 for the treatment of SMA patients irrespective of age and disease severity. Most data on therapeutic efficacy are available for the infantile-onset SMA. For patients with SMA type 2 and type 3, there is still a lack of sufficient evidence and long-term experience for nusinersen treatment. Here, we report data from the SMArtCARE registry of non-ambulant children with SMA type 2 and typen 3 under nusinersen treatment with a follow-up period of up to 38 months. METHODS: SMArtCARE is a disease-specific registry with data on patients with SMA irrespective of age, treatment regime or disease severity. Data are collected during routine patient visits as real-world outcome data. This analysis included all non-ambulant patients with SMA type 2 or 3 below 18 years of age before initiation of treatment. Primary outcomes were changes in motor function evaluated with the Hammersmith Functional Motor Scale Expanded (HFMSE) and the Revised Upper Limb Module (RULM). RESULTS: Data from 256 non-ambulant, pediatric patients with SMA were included in the data analysis. Improvements in motor function were more prominent in upper limb: 32.4% of patients experienced clinically meaningful improvements in RULM and 24.6% in HFMSE. 8.6% of patients gained a new motor milestone, whereas no motor milestones were lost. Only 4.3% of patients showed a clinically meaningful worsening in HFMSE and 1.2% in RULM score. CONCLUSION: Our results demonstrate clinically meaningful improvements or stabilization of disease progression in non-ambulant, pediatric patients with SMA under nusinersen treatment. Changes were most evident in upper limb function and were observed continuously over the follow-up period. Our data confirm clinical trial data, while providing longer follow-up, an increased number of treated patients, and a wider range of age and disease severity.

Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming.

Hepadnaviruses, including human hepatitis B virus (HBV), replicate their tiny DNA genomes by protein-primed reverse transcription of a pregenomic (pg) RNA. Replication initiation as well as pgRNA encapsidation depend on the interaction of the viral polymerase, P protein, with the ε RNA element, featuring a lower and an upper stem, a central bulge, and an apical loop. The bulge, somehow assisted by the loop, acts as template for a P protein-linked DNA oligo that primes full-length minus-strand DNA synthesis. Phylogenetic conservation and earlier mutational studies suggested the highly based-paired ε structure as crucial for productive interaction with P protein. Using the tractable duck HBV (DHBV) model we here interrogated the entire apical DHBV ε (Dε) half for sequence- and structure-dependent determinants of in vitro priming activity, replication, and, in part, in vivo infectivity. This revealed single-strandedness of the bulge, a following G residue plus the loop subsequence GUUGU as the few key determinants for priming and initiation site selection; unexpectedly, they functioned independently of a specific structure context. These data provide new mechanistic insights into avihepadnaviral replication initiation, and they imply a new concept towards a feasible in vitro priming system for human HBV.

Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo.

Chronic infection with hepatitis B virus (HBV), a small DNA virus that replicates by reverse transcription of a pregenomic (pg) RNA precursor, greatly increases the risk for terminal liver disease. RNA interference (RNAi) based therapy approaches have shown potential to overcome the limited efficacy of current treatments. However, synthetic siRNAs as well as small hairpin (sh) RNAs expressed from non-integrating vectors require repeated applications; integrating vectors suffer from safety concerns. We pursue a new concept by which HBV itself is engineered into a conditionally replicating, wild-type HBV dependent anti-HBV shRNA vector. Beyond sharing HBV's hepatocyte tropism, such a vector would be self-renewing, but only as long as wild-type HBV is present. Here, we realized several important aspects of this concept. We identified two distinct regions in the 3.2 kb HBV genome which tolerate replacement by shRNA expression cassettes without compromising reverse transcription when complemented in vitro by HBV helper constructs or by wild-type HBV; a representative HBV shRNA vector was infectious in cell culture. The vector-encoded shRNAs were active, including on HBV as target. A dual anti-HBV shRNA vector delivered into HBV transgenic mice, which are not susceptible to HBV infection, by a chimeric adenovirus-HBV shuttle reduced serum hepatitis B surface antigen (HBsAg) up to ∼4-fold, and virus particles up to ∼20-fold. Importantly, a fraction of the circulating particles contained vector-derived DNA, indicating successful complementation in vivo. These data encourage further investigations to prove antiviral efficacy and the predicted self-limiting vector spread in a small animal HBV infection model.