Clinically relevant dosing and pharmacokinetics of DNA-encoded antibody therapeutics in a sheep model.

DNA-encoded delivery and in vivo expression of antibody therapeutics presents an innovative alternative to conventional protein production and administration, including for cancer treatment. To support clinical translation, we evaluated this approach in 18 40-45 kg sheep, using a clinical-matched intramuscular electroporation (IM EP) and hyaluronidase-plasmid DNA (pDNA) coformulation setup. Two cohorts of eight sheep received either 1 or 4 mg pDNA encoding an ovine anti-cancer embryonic antigen (CEA) monoclonal antibody (mAb; OVAC). Results showed a dose-response with average maximum serum concentrations of respectively 0.3 and 0.7 µg/ml OVAC, 4-6 weeks after IM EP. OVAC was detected in all 16 sheep throughout the 6-week follow-up, and no anti-OVAC antibodies were observed. Another, more exploratory, cohort of two sheep received a 12 mg pOVAC dose. Both animals displayed a similar dose-dependent mAb increase and expression profile in the first two weeks. However, in one animal, an anti-OVAC antibody response led to loss of mAb detection four weeks after IM EP. In the other animal, no anti-drug antibodies were observed. Serum OVAC concentrations peaked at 4.9 µg/ml 6 weeks after IM EP, after which levels gradually decreased but remained detectable around 0.2 to 0.3 µg/ml throughout a 13-month follow-up. In conclusion, using a delivery protocol that is currently employed in clinical Phase 1 studies of DNA-based antibodies, we achieved robust and prolonged in vivo production of anti-cancer DNA-encoded antibody therapeutics in sheep. The learnings from this large-animal model regarding the impact of pDNA dose and host immune response on the expressed mAb pharmacokinetics can contribute to advancing clinical translation.

A DNA-Launched Nanoparticle Vaccine Elicits CD8+ T-cell Immunity to Promote In Vivo Tumor Control.

Cytolytic T cells (CTL) play a pivotal role in surveillance against tumors. Induction of CTL responses by vaccination may be challenging, as it requires direct transduction of target cells or special adjuvants to promote cross-presentation. Here, we observed induction of robust CTL responses through electroporation-facilitated, DNA-launched nanoparticle vaccination (DLnano-vaccines). Electroporation was observed to mediate transient tissue apoptosis and macrophage infiltration, which were deemed essential to the induction of CTLs by DLnano-vaccines through a systemic macrophage depletion study. Bolus delivery of protein nano-vaccines followed by electroporation, however, failed to induce CTLs, suggesting direct in vivo production of nano-vaccines may be required. Following these observations, new DLnano-vaccines scaffolding immunodominant melanoma Gp100 and Trp2 epitopes were designed and shown to induce more potent and consistent epitope-specific CTL responses than the corresponding DNA monomeric vaccines or CpG-adjuvanted peptide vaccines. DNA, but not recombinant protein, nano-vaccinations induced CTL responses to these epitopes and suppressed melanoma tumor growth in mouse models in a CD8+ T-cell-dependent fashion. Further studies to explore the use of DLnano-vaccines against other cancer targets and the biology with which they induce CTLs are important.

In Vivo Assembly of Nanoparticles Achieved through Synergy of Structure-Based Protein Engineering and Synthetic DNA Generates Enhanced Adaptive Immunity.

Nanotechnologies are considered to be of growing importance to the vaccine field. Through decoration of immunogens on multivalent nanoparticles, designed nanovaccines can elicit improved humoral immunity. However, significant practical and monetary challenges in large-scale production of nanovaccines have impeded their widespread clinical translation. Here, an alternative approach is illustrated integrating computational protein modeling and adaptive electroporation-mediated synthetic DNA delivery, thus enabling direct in vivo production of nanovaccines. DNA-launched nanoparticles are demonstrated displaying an HIV immunogen spontaneously self-assembled in vivo. DNA-launched nanovaccines induce stronger humoral responses than their monomeric counterparts in both mice and guinea pigs, and uniquely elicit CD8+ effector T-cell immunity as compared to recombinant protein nanovaccines. Improvements in vaccine responses recapitulate when DNA-launched nanovaccines with alternative scaffolds and decorated antigen are designed and evaluated. Finally, evaluation of functional immune responses induced by DLnanovaccines demonstrates that, in comparison to control mice or mice immunized with DNA-encoded hemagglutinin monomer, mice immunized with a DNA-launched hemagglutinin nanoparticle vaccine fully survive a lethal influenza challenge, and have substantially lower viral load, weight loss, and influenza-induced lung pathology. Additional study of these next-generation in vivo-produced nanovaccines may offer advantages for immunization against multiple disease targets.

In vivo delivery of synthetic DNA-encoded antibodies induces broad HIV-1-neutralizing activity.

Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed that possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities, especially for multiantibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed, globally affecting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dmAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dmAb cassettes and studied their activity in small and large animals. Sera from animals administered dmAbs neutralized multiple HIV-1 isolates with activity similar to that of their parental recombinant mAbs. Delivery of multiple dmAbs to a single animal led to increased neutralization breadth. Two dmAbs, PGDM1400 and PGT121, were advanced into nonhuman primates for study. High peak-circulating levels (between 6 and 34 µg/ml) of these dmAbs were measured, and the sera of all animals displayed broad neutralizing activity. The dmAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.

Delayed diagnosis of spontaneous intestinal perforation among very low birth weight neonates: A single center experience.

OBJECTIVE: To assess incidence and effect of delayed diagnosis of spontaneous intestinal perforation (SIP). STUDY DESIGN: Retrospective case series review of 58 VLBW neonates with SIP at our institution. RESULT: SIP was diagnosed in 6.1%, 10%, and 15.1% of VLBW, ELBW, and ≤750 g neonates, respectively. Abdominal distension (58.6%) and abdominal discoloration (53.4%) were the most common presenting signs/symptoms. Smaller (≤750 g) neonates were more likely to present with hypotension and higher FiO2, and larger (751-1500 g) neonates with increased abdominal girth and abdominal distension. All but one neonate had radiographic pneumoperitoneum, and 25.9% had pneumoperitoneum on an X-ray prior to the X-ray at SIP diagnosis. An education module reduced delay in SIP diagnosis. CONCLUSION: SIP presentation varies by birth weight and gestational age. Since SIP diagnosis is often first suggested on X-ray, all X-rays of VLBW neonates in the first 2 weeks of life should be scrutinized for pneumoperitoneum.

Variation in management of in-hospital newborn falls: a single-center experience.

OBJECTIVE There are only 3 small case series in the literature that report on the management of in-hospital newborn falls (NFs), and recommendations are unclear. The authors performed a retrospective review to determine outcome and differences in management and to understand why management of NFs varies at their institution. METHODS All NFs occurring within the authors' institution over a 3.5-year period were reviewed. Post-fall management and outcomes of each incident were compared. RESULTS There were 24 NFs out of 40,349 deliveries (5.9 NFs/10,000 deliveries). The mechanism of injury was nearly identical in 22 of 24 falls (the newborn fell to the floor from a parent in a bed or chair), and physical examination findings were normal or benign in all cases. Unexplained management variation based solely on clinician preference was noted, including observation only (in 13 cases), skull radiograph (in 7), head CT scan (in 6), bone survey (in 4), and head ultrasound examination (in 1), with some babies having more than 1 study. Two babies had nondepressed linear parietal fractures diagnosed by skull radiograph, and 2 babies had small subdural hemorrhages diagnosed by head CT scan. All 24 babies had normal findings on examination at discharge. CONCLUSIONS There is a high incidence of nondepressed linear parietal skull fractures associated with NFs. However, since associated intracranial injury is uncommon, imaging studies may not be routinely performed. Neonatal intensive care unit admission, head CT, and neurosurgical evaluation are reserved for the rare baby with abnormal physical examination or neurological findings.

Multifunctional poly(ß-amino ester) hydrogel microparticles in periodontal in situ forming drug delivery systems.

In situ forming implants (ISIs) formed from poly(lactic-co-glycolic acid) (PLGA) have been commercialized for local drug delivery to treat periodontitis, but drug release from these bulk materials is typically subject to an initial burst. In addition, PLGA has inferior material properties for the dynamic mechanical environment of gingival tissue. In this work, poly(ß-amino ester) (PBAE) hydrogel microparticles were incorporated into a PLGA matrix to provide several new functions: mechanical support, porosity, space-filling, and controlled co-delivery of antimicrobial and osteogenic drugs. First, the effects of PBAE microparticles on ISI architecture and material properties throughout degradation were investigated. Second, the influence of PBAE microparticles on drug release kinetics was quantified. Over a 15 d period, ISIs containing PBAE microparticles possessed greater porosity, ranging from 42-80%, compared to controls, which ranged from 24-54% (p < 0.001), and these ISIs also developed significantly greater accessible volume to simulated cell-sized spheres after 5 d or more of degradation (p < 0.001). PBAE-containing ISIs possessed a more uniform microarchitecture, which preserved mechanical resilience after cyclical loading (p < 0.001), and the materials swelled to fill the injected space, which significantly increased interfacial strength in an artificial periodontal pocket (p < 0.0001). PBAE microparticles eliminated the burst of freely-mixed simvastatin compared to 36% burst from controls (p < 0.0001), and high-dose doxycycline release was prolonged from 2 d to 7 d by pre-loading drug into the microparticles. PBAE-containing PLGA ISIs are more effective space-filling scaffolds and offer improved release kinetics compared to existing ISIs used to treat periodontitis.

Synergistic local drug delivery in a piglet model of ischemic osteonecrosis: a preliminary study.

A locally injectable system sequentially delivering an antiresorptive drug (clodronate) followed by an osteogenic agent (simvastatin) was hypothesized to improve femoral head microarchitecture, size, and shape compared with untreated or partial treatment groups in an established piglet osteonecrosis model. After 6 weeks, the clodronate+simvastatin treatment resulted in no collapse, microCT measurements and epiphyseal quotients within 10% of control, normal microstructure, and healthy histology. All other groups exhibited collapse, lower epiphyseal quotients and total femoral head volumes (P<0.05), and abnormal histology. This pilot study provides evidence of synergistic antiresorptive and osteogenic activities, which may prevent femoral head collapse in Perthes disease.

Effects of epidermal growth factor-loaded mucoadhesive films on wounded oral tissue rafts.

Current treatments for traumatic oral mucosal wounds include the gold standard of autologous tissue and alternative tissue-engineered grafts. While use of autografts has disadvantages of minimal availability of oral keratinized tissue, second surgery, and donor site discomfort, tissue-engineered grafts are limited by their unavailability as off-the-shelf products owing to their fabrication time of 4-8 weeks. Hence, the current work aimed to develop a potentially cost-effective, readily available device capable of enhancing native mucosal regeneration. Considering the key role of epidermal growth factor (EGF) in promoting mucosal wound regeneration and the advantages of mucoadhesive delivery systems, mucoadhesive films composed of polyvinylpyrrolidone and carboxymethylcellulose were developed to provide sustained release of EGF for a minimum of 6 h. Bioactivity of released EGF supernatants was then confirmed by its ability to promote proliferation of BALB/3T3 fibroblasts. Efficacy of the developed system was then investigated in vitro using buccal tissues (ORL 300-FT) as a potential replacement for small animal studies. Although the mucoadhesive films achieved their desired role of delivering bioactive EGF in a sustained manner, treatment with EGF, irrespective of its release from the films or solubilized in medium, caused a hyperparakeratotic response from in vitro tissues with distinguishable histological features including thickening of the spinous layer, intra- and intercellular edema, and pyknotic nuclei. These significant morphological changes were associated with no improvements in wound closure. These observations raise questions about the potential of using in vitro tissues as a wound healing model and substitute for small animal studies. The mucoadhesive delivery system developed, however, with its potential for sustained release of bioactive growth factors and small molecules, may be loaded with other desired compounds, with or without EGF, to accelerate the process of wound healing.

Hydroxyapatite-reinforced in situ forming PLGA systems for intraosseous injection.

In situ forming poly(lactic-co-glycolic acid) (PLGA) implants have not been strongly considered for bone applications because of their poor mechanical properties. Here, in situ forming scaffolds containing hydroxyapatite micro- and nanoparticles were characterized to determine their mechanical properties, injectability, and microarchitecture. Scaffolds were prepared with various concentrations of hydroxyapatite, as well as poly(ß-amino ester) microparticles that facilitate drug delivery. Strength was increased threefold, from 2 to 6 MPa, while compressive modulus was improved sixfold, from 24 to 141 MPa, via the addition of 30% nanohydroxyapatite, which provided greater benefits at equivalent concentrations compared to micro-hydroxyapatite. Scaffolds retained a uniformly porous microarchitecture, and hydroxyapatite particles were distributed evenly throughout the PLGA phase. Injectability, determined by the force required to inject 0.5 mL of material within 60 s, remained clinically acceptable at <50 N at 30% w/w hydroxyapatite and up to 10% w/w PBAE microparticles. Ex vivo injections into intact porcine femoral heads increased compressive modulus of trabecular bone from 81 to 180 MPa and strength from 3.5 to 5.9 MPa. This injectable scaffold offers mechanical reinforcement coupled with previously demonstrated drug delivery potential in a single injection for bone-weakening conditions, such as osteonecrosis or osteoporosis.

Improved small molecule drug release from in situ forming poly(lactic-co-glycolic acid) scaffolds incorporating poly(ß-amino ester) and hydroxyapatite microparticles.

In situ forming implants are an attractive choice for controlled drug release into a fixed location. Currently, rapidly solidifying solvent exchange systems suffer from a high initial burst, and sustained release behavior is tied to polymer precipitation and degradation rate. The present studies investigated addition of hydroxyapatite (HA) and drug-loaded poly(ß-amino ester) (PBAE) microparticles to in situ forming poly(lactic-co-glycolic acid) (PLGA)-based systems to prolong release and reduce burst. PBAEs were synthesized, imbibed with simvastatin (osteogenic) or clodronate (anti-resorptive), and then ground into microparticles. Microparticles were mixed with or without HA into a PLGA solution, and the mixture was injected into buffer, leading to precipitation and creating solid scaffolds with embedded HA and PBAE microparticles. Simvastatin release was prolonged through 30 days, and burst release was reduced from 81 to 39% when loaded into PBAE microparticles. Clodronate burst was reduced from 49 to 32% after addition of HA filler, but release kinetics were unaffected after loading into PBAE microparticles. Scaffold dry mass remained unchanged through day 15, with a pronounced increase in degradation rate after day 30, while wet scaffolds experienced a mass increase through day 25 due to swelling. Porosity and pore size changed throughout degradation, likely due to a combination of swelling and degradation. The system offers improved release kinetics, multiple release profiles, and rapid solidification compared to traditional in situ forming implants.