Adeno-associated viral (AAV) vectors have traditionally been viewed as predominantly nonintegrating, with limited concerns for oncogenesis. However, accumulating preclinical data have shown that AAV vectors integrate more often than previously appreciated, with the potential for genotoxicity. To understand the consequences of AAV vector integration, vigilance for rare genotoxic events after vector administration is essential. Here, we investigate the development of multicentric lymphoma in a privately owned dog, PC9, with severe hemophilia A that was treated with an AAV8 vector encapsidating a B domain-deleted canine coagulation F8 gene. PC9 developed an aggressive B cell lineage multicentric lymphoma 3.5 years after AAV treatment. Postmortem analysis of the liver, spleen, and lymph nodes showed the expected biodistribution of the AAV genome. Integration events were found both in PC9 and a second privately owned hemophilia A dog treated similarly with canine F8 gene transfer, which died of a bleeding event without evidence of malignancy. However, we found no evidence of expanded clones harboring a single integration event, indicating that AAV genome integrations were unlikely to have contributed to PC9's cancer. These findings suggest AAV integrations occur but are mostly not genotoxic and support the safety profile of AAV gene therapy.

Background: Current AAV gene therapy for hemophilia A (HA) is limited by low sustained factor VIII (FVIII) levels. In contrast, AAV gene therapy for hemophilia B benefits from the widespread adoption of the single amino acid (AA) substituted high-specific-activity FIX variant R338L. We have previously shown that wild-type (WT) FIX has not evolved for maximal specific activity, but rather has evolutionary conserved AAs that limit procoagulant activity (Samelson-Jones et al. Blood Advances 2021). FVIII may have evolved with similar considerations. Based on this work, we developed a novel screening technique, termed Evolution Directed Gene Engineering (EDGE), to identify new hyperactive single AA substituted FVIII variant transgenes to address the current limitations of HA gene therapy. Aims: To find and characterize single AA substituted FVIII variants that can enhance gene transfer. Methods: EDGE evaluates AA positions fulfilling 2 criteria: 1) highly evolutionary conserved and 2) not associated with the disease phenotype. Here, EDGE compared 33 mammalian FVIII orthologues and then eliminated positions associated with known HA-causing missense mutations. FVIII variants were transiently expressed in Huh7 cells and cultured media was assayed for FVIII activity and antigen. Recombinant FVIII protein of lead candidates were purified to homogeneity and assayed for FVIII activity with one- and two-stage aPTT assays, thrombin generation assay (TGA), and enzyme kinetics studies. The in vivo efficacy of the variants was tested by tail clip assays of HA mice after retro-orbital delivery of the protein or liver-directed AAV gene therapy. Results: Out of 52 AA positions identified by EDGE, we found 12 AA positions in FVIII where single AA substitutions increased specific activity ≥2-fold (Figure 1A). The largest increase in specific activity was 6-fold, which occurred for multiple AA substitutions at position 659. Purified recombinant protein of FVIII-K659M and -K659V both demonstrated 6-fold increased activity in one- and two-stage aPTT assays, but comparable thrombin activation and A2-stability to FVIII-WT. TGA show the FVIII-K659M/V variants have greater than 6-fold improvements in endogenous thrombin potential and peak thrombin generation. Compared to FVIII-WT, FVIII-K659M/V proteins have an increased k cat and decreased appK D which suggests that the mechanism of increased specific activity is due to enhanced interactions with FIXa. This is similar to the mechanism of hyperactivity of FIX-R338L. FVIII-K659M/V also demonstrated enhanced procoagulant activity in vivo. In tail clip injuries of HA mice (n≥4) treated with purified protein, the FVIII-K659M/V proteins restored normal hemostasis at 5-fold lower concentrations than FVIII-WT. When mice were treated with equal doses of 2.5 µg/kg of protein, FVIII-K659M/V restored normal hemostasis while FVIII-WT had no significant change in blood loss (Figure 1B). As expected, 12.5 µg/kg of FVIII-WT protein was required to restore blood loss to WT mouse levels. FVIII-K659 substituted variants also enhanced AAV based gene therapy. HA mice (n=6) treated with AAV8 vectors expressing FVIII-K659C had 6-fold higher plasma FVIII activity than mice (n=6) treated at the same dose with AAV8 vectors expressing FVIII-WT. HA mice that received 4 x 10 12 vg/kg of AAV8 FVIII-K659C had less bleeding than HA mice that received the same dose of AAV8 FVIII-WT, but similar blood loss as WT mice after tail clip injury. Conclusions: The success of EDGE in identifying multiple new high specific activity FVIII variants supports the hypothesis that FVIII-WT is not optimized for specific activity and thus is amenable to bioengineering approaches. The ≥5-fold enhanced in vitro and in vivo hemostatic activity of the FVIII-K659 substituted variants and initial murine gene therapy studies support their translational potential to address current limitations of HA gene therapy. These results also implicate K659 in modulating the FVIIIa/FIXa interaction. EDGE may also be applicable to other gene therapy targets as well.

Gene delivery to fertilized eggs is often the first step in creation of transgenic animals, CRISPR knock-out, or early developmental studies. The zona pellucida, a hardened glycoprotein matrix surrounding the mammalian fertilized eggs, often complicates gene delivery by forming a barrier against transfection reagents and viruses. High efficiency techniques to perforate or penetrate the zona allow for access and gene delivery to fertilized eggs. However, these techniques often rely on highly skilled technologists, are costly, and require specialized equipment for micromanipulation, laser perforation, or electroporation. Here, we report that adenoassociated viruses (AAVs) with serotypes 1 or DJ can efficiently diffuse across the zona to deliver genes without any manipulations to fertilized eggs. We observe lowered rates of embryo development after treatment of embryos with all AAV serotypes. However, we were able to reduce adverse effects on embryo development by exposing embryos to AAVs at later stages of in vitro development. AAVs have low immune response and do not incorporate into their host chromosomes to cause insertional mutations. Hence, AAVs can serve as a highly effective tool for transient delivery of genes to fertilized mammalian eggs.

Significant concerns have arisen over the past 3 y from the increased global spread of the mosquito-borne flavivirus, Zika. Accompanying this spread has been an increase in cases of the devastating birth defect microcephaly as well as of Guillain–Barré syndrome in adults in many affected countries. Currently there is no vaccine or therapy for this infection; however, we sought to develop a combination approach that provides more rapid and durable protection than traditional vaccination alone. A novel immune-based prophylaxis/therapy strategy entailing the facilitated delivery of a synthetic DNA consensus prME vaccine along with DNA-encoded anti-ZIKV envelope monoclonal antibodies (dMAb) were developed and evaluated for antiviral efficacy. This immediate and persistent protection strategy confers the ability to overcome shortcomings inherent with conventional active vaccination or passive immunotherapy. A collection of novel dMAbs were developed which were potent against ZIKV and could be expressed in serum within 24–48 h of in vivo administration. The DNA vaccine, from a previous development, was potent after adaptive immunity was developed, protecting against infection, brain and testes pathology in relevant mouse challenge models and in an NHP challenge. Delivery of potent dMAbs protected mice from the same murine viral challenge within days of delivery. Combined injection of dMAb and the DNA vaccine afforded rapid and long-lived protection in this challenge model, providing an important demonstration of the advantage of this synergistic approach to pandemic outbreaks.

BackgroundVariants inPRPF31, which encodes pre-mRNA processing factor 31 homolog, are known to cause autosomal-dominant retinitis pigmentosa (adRP) with incomplete penetrance. However, the majority of mutations cause null alleles, with only two proven pathogenic missense mutations. We identified a novel missense mutation inPRPF31in a family with adRP.MethodsWe performed whole exome sequencing to identify possible pathogenic mutations in the proband of a family with adRP. Available affected family members had a full ophthalmological evaluation including kinetic and two-colour dark adapted static perimetry, electroretinography and multimodal imaging of the retina. Two patients had evaluations covering nearly 20 years. We carried out segregation analysis of the probable mutation,PRPF31c.590T>C. We evaluated the cellular localisation of the PRPF31 variant (p.Leu197Pro) compared with the wildtype PRPF31 protein.ResultsPRPF31c.590T>C segregated with the disease in this four-generation autosomal dominant pedigree. There was intrafamilial variability in disease severity. Nyctalopia and mid-peripheral scotomas presented from the second to the fourth decade of life. There was severe rod >cone dysfunction. Visual acuity (VA) was relatively intact and was maintained until later in life, although with marked interocular asymmetries. Laboratory studies showed that the mutant PRPF31 protein (p.Leu197Pro) does not localise to the nucleus, unlike the wildtype PRPF31 protein. Instead, mutant protein resulted in punctate localisation to the cytoplasm.Conclusionsc.590T>C is a novel pathogenic variant inPRPF31causing adRP with incomplete penetrance. Disease may be due to protein misfolding and associated abnormal protein trafficking to the nucleus.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Lentiviruses are highly efficient vehicles for delivering genes into cells. They readily transduce primary and immortalized cells in vivo and in vitro. Genes delivered by lentiviruses are incorporated and replicated as part of their host genome and therefore offer a powerful tool for creation of stable cell lines and transgenic animals. However, the zona pellucida surrounding the fertilized eggs acts as a barrier and hinders lentiviral transduction of embryos. Here, we utilize a laser, typically used to perforate the zona pellucida for in vitro fertilization, to permeabilize the zona for lentiviral gene delivery. A single hole in the zona is sufficient for the lentivirus to gain access to fertilized eggs without the need for microinjection for en masse gene delivery. Embryos generated by this method elicit no damage and can develop to term for creation of transgenic animals.