Generation of enhanced gene delivery vectors by directed evolution of adeno-associated virus

Despite promising advance in the development of viral vectors based on AAV for human gene therapy, several major hurdles for a more general use remain. Among these, efficient in vivo applications are limited by the high prevalence of neutralizing antibodies in the human population, which can reduce or eliminate transgene expression. A successful prevention of antibody-mediated vector neutralisation requires the modification of specific epitopes of the viral capsid responsible for Ab binding. The aim of this work was to demonstrate that immune-escaping capsid variants can be generated through genetic modifications of the virus by taking advantage of combinatorial engineering and directed evolution protocols. A library of 107 AAV mutants carrying random point mutations scattered throughout the capsid gene of AAV was created by error prone PCR and screened for clones that were able to avoid neutralization by AAV-neutralizing human sera. Three mutants carrying the mutations R459G, R459K and N551D respectively and a double mutant with a combined R459K/N551D mutation were strongly enriched after the selection procedure. Characterisation of these clones showed an immune-escaping phenotype for all mutants. However, the combination mutant proved to be superior in both evasion of neutralization and infectivity, leading to the assumption that multiple mutations convey enhanced effects. Therefore, the remaining pool was subjected to DNA shuffling and additional error prone PCR, yielding a second-generation library, which was screened for further improved phenotypes. In this context, a method which we called evolution monitoring was devised allowing optimization of several experimental conditions that are typically critical for successful outcome of library panning. These refinements yielded novel variants with further enhanced immune-escape abilities and infectivity in comparison to previously selected mutants. Finally, obtained data suggests an enormous potential for using the here developed tools to study infection biology of viruses by reverse genetics. This work showed for the first time that error prone PCR and DNA shuffling can be successfully applied for genetic engineering of a virus by a directed evolution approach. In principle, using appropriate selection protocols these techniques should be adaptable for addressing a wide variety of challenges concerning AAV in particular and virology in general

Activity of tafasitamab in combination with rituximab in subtypes of aggressive lymphoma

BackgroundDespite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects.MethodsAntibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL).ResultsThree different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model.ConclusionThis study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo

Full-wedge metallic reconstruction of glenoid bone deficiency in reverse shoulder arthroplasty

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Medial epicondylitis is a common orthopedic condition that typically results from overuse or previous microtrauma of the flexor-pronator mass. Repetitive eccentric loading of the muscles leads to subsequent degeneration of the flexor tendons.</jats:p> </jats:sec><jats:sec> <jats:title>Diagnosis</jats:title> <jats:p>Patients present with a painful elbow. In the case of concomitant elbow pathologies, including ulnar neuritis and ulnar collateral ligament injury, there should be a detailed examination. Generally, the diagnosis is based on the clinical examination. T2-weighted magnetic resonance imaging can be useful for chronic courses, over 6 months.</jats:p> </jats:sec><jats:sec> <jats:title>Treatment</jats:title> <jats:p>Nonsurgical management is the mainstay of treatment. Hence, surgical treatment may be indicated for patients with persistent symptoms after conservative treatment. In the case of a surgical treatment, arthroscopy can be useful to capture concomitant elbow pathologies.</jats:p> </jats:sec&gt

Reverse Shoulder Arthroplasty for Proximal Humerus Head-Split Fractures—A Retrospective Cohort Study

Head-split fractures are proximal humerus fractures (PHF) that result from fracture lines traversing the articular surface. While head-split fractures are rare, surgical treatment of these complex injuries can be extremely challenging and is associated with high rates of complications. Treatment using primary reverse shoulder arthroplasty (RSA) has been associated with moderate complication rates and reproducible clinical results. The aim of this study was to evaluate clinical and radiographic outcomes, and complication rates of RSA for head-split PHF. Twenty-six patients were evaluated based on Constant Score (CS) and range of motion of both shoulders and Subjective Shoulder Value (SSV). Radiographic analysis evaluated tuberosity healing, prosthetic loosening and scapular notching. Patients achieved good clinical results with a CS of 73.7 points and SSV of 82% after a mean follow-up of 50 months. The relative CS comparing operated versus the unaffected shoulder was 92%. Greater tuberosity healing was achieved in 61%. Patients who suffered a high-energy trauma reached a significantly greater functional outcome. Patients who suffered multifragmentation to the humeral head performed the worst. There were no cases of loosening; scapular notching was visible in two cases. The complication rate was 8%. RSA is an adequate treatment option with for head-split PHF in elderly patients

Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking

To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry

Heparan Sulfate Proteoglycan Binding Properties of Adeno-Associated Virus Retargeting Mutants and Consequences for Their In Vivo Tropism

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues