Humanization and functional characterization of enhanced coagulation factor IX variants identified through ancestral sequence reconstruction.

BACKGROUND: Laboratory resurrection of ancient coagulation factor (F) IX variants generated through ancestral sequence reconstruction led to the discovery of a FIX variant, designated An96, which possesses enhanced specific activity independent of and additive to that provided by human p.Arg384Lys, referred to as FIX-Padua. OBJECTIVES: The goal of the current study was to identify the amino acid substitution(s) responsible for the enhanced activity of An96 and create a humanized An96 FIX transgene for gene therapy application. METHODS: Reductionist screening approaches, including domain swapping and scanning residue substitution, were used and guided by one-stage FIX activity assays. In vitro characterization of top candidates included recombinant high-purity preparation, specific activity determination, and enzyme kinetic analysis. Final candidates were packaged into adeno-associated viral (AAV) vectors and delivered to hemophilia B mice. RESULTS: Five of 42 total amino acid substitutions in An96 appear sufficient to retain the enhanced activity of An96 in an otherwise human FIX variant. Additional substitution of the Padua variant further increased the specific activity 5-fold. This candidate, designated ET9, demonstrated 51-fold greater specific activity than hFIX. AAV2/8-ET9 treated hemophilia B mice produced plasma FIX activities equivalent to those observed previously for AAV2/8-An96-Padua, which were 10-fold higher than AAV2/8-hFIX-Padua. CONCLUSION: Starting from computationally inferred ancient FIX sequences, novel amino acid substitutions conferring activity enhancement were identified and translated into an AAV-FIX gene therapy cassette demonstrating high potency. This ancestral sequence reconstruction discovery and sequence mapping refinement approach represents a promising platform for broader protein drug and gene therapy candidate optimization.

Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function.

Cellular therapies are currently employed to treat a variety of disease processes. For T cell-based therapies, success often relies on the metabolic fitness of the T cell product, where cells with enhanced metabolic capacity demonstrate improved in vivo efficacy. AMP-activated protein kinase (AMPK) is a cellular energy sensor which combines environmental signals with cellular energy status to enforce efficient and flexible metabolic programming. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates, higher maximal oxygen consumption rate, and augmented spare respiratory capacity. These changes were accompanied by increased proliferation and inflammatory cytokine production, particularly within restricted glucose environments. Introduction of AMPKγ2 into bulk CD4 T cells decreased RNA expression of canonical Th2 genes, including the cytokines interleukin (IL)-4 and IL-5, while introduction of AMPKγ2 into individual Th subsets universally favored proinflammatory cytokine production and a downregulation of IL-4 production in Th2 cells. When AMPKγ2 was overexpressed in regulatory T cells, both in vitro proliferation and suppressive capacity increased. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and functional potential of human T cells for use in a variety of adoptive cellular therapies.

Ligand-based targeting of c-kit using engineered γδ T cells as a strategy for treating acute myeloid leukemia.

The application of immunotherapies such as chimeric antigen receptor (CAR) T therapy or bi-specific T cell engager (BiTE) therapy to manage myeloid malignancies has proven more challenging than for B-cell malignancies. This is attributed to a shortage of leukemia-specific cell-surface antigens that distinguish healthy from malignant myeloid populations, and the inability to manage myeloid depletion unlike B-cell aplasia. Therefore, the development of targeted therapeutics for myeloid malignancies, such as acute myeloid leukemia (AML), requires new approaches. Herein, we developed a ligand-based CAR and secreted bi-specific T cell engager (sBite) to target c-kit using its cognate ligand, stem cell factor (SCF). c-kit is highly expressed on AML blasts and correlates with resistance to chemotherapy and poor prognosis, making it an ideal candidate for which to develop targeted therapeutics. We utilize γδ T cells as a cytotoxic alternative to αß T cells and a transient transfection system as both a safety precaution and switch to remove alloreactive modified cells that may hinder successful transplant. Additionally, the use of γδ T cells permits its use as an allogeneic, off-the-shelf therapeutic. To this end, we show mSCF CAR- and hSCF sBite-modified γδ T cells are proficient in killing c-kit+ AML cell lines and sca-1+ murine bone marrow cells in vitro. In vivo, hSCF sBite-modified γδ T cells moderately extend survival of NSG mice engrafted with disseminated AML, but therapeutic efficacy is limited by lack of γδ T-cell homing to murine bone marrow. Together, these data demonstrate preclinical efficacy and support further investigation of SCF-based γδ T-cell therapeutics for the treatment of myeloid malignancies.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers.

Adoptive cell therapy (ACT) utilizing γδ T cells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific T cell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ T cells with CAR or secreted bispecific T cell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ T cells. Using a CD19-specific CAR, approximately 60% of γδ T cells are modified after mRNA electroporation and these cells show potent anticancer activity in vitro and in vivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ T cell cytotoxicity, both in vitro and in vivo, and promotes killing of target cells by modified and unmodified γδ T cells. Taken together, we show that transient transfection of γδ T cells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.

Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation.

NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.

Coagulation factor IX gene transfer to non-human primates using engineered AAV3 capsid and hepatic optimized expression cassette.

Hepatic gene transfer with adeno-associated viral (AAV) vectors shows much promise for the treatment of the X-linked bleeding disorder hemophilia B in multiple clinical trials. In an effort to further innovate this approach and to introduce alternative vector designs with potentially superior features into clinical development, we recently built a vector platform based on AAV serotype 3 because of its superior tropism for human hepatocytes. A vector genome with serotype-matched inverted terminal repeats expressing hyperactive human coagulation factor IX (FIX)-Padua was designed for clinical use that is optimized for translation using hepatocyte-specific codon-usage bias and is depleted of immune stimulatory CpG motifs. Here, this vector genome was packaged into AAV3 (T492V + S663V) capsid for hepatic gene transfer in non-human primates. FIX activity within or near the normal range was obtained at a low vector dose of 5 × 1011 vector genomes/kg. Pre-existing neutralizing antibodies, however, completely or partially blocked hepatic gene transfer at that dose. No CD8+ T cell response against capsid was observed. Antibodies against the human FIX transgene product formed at a 10-fold higher vector dose, albeit hepatic gene transfer was remarkably consistent, and sustained FIX activity in the normal range was nonetheless achieved in two of three animals for the 3-month duration of the study. These results support the use of this vector at low vector doses for gene therapy of hemophilia B in humans.

Bathroom Modifications, Clutter, and Tripping Hazards: Prevalence and Changes after Incident Falls in Community-Dwelling Older Adults.

Objectives: To describe the prevalence of bathroom modifications, clutter, and tripping hazards in the homes of US older adults and to examine changes after an incident fall. Methods: We used data from the 2015-2017 National Health and Aging Trends Study (n = 7499). Outcomes were the prevalence of bathroom modifications, clutter, and tripping hazards and changes after incident fall. Results: In 2015, 26.5% of community-dwelling older adults had clutter or tripping hazards in the home, and 69.3% had at least one bathroom modification. Compared to nonfallers, older adults with multiple falls were significantly more likely to modify the bathroom. The magnitude of hazard reduction was similar after multiple falls but was not statistically significant. Discussion: Bathroom modifications are common and increase after multiple falls. A single incident fall does not appear to lead to home environment changes to reduce fall risk. Targeting home hazards may be an opportunity to reduce fall risk.

Development of a Clinical Candidate AAV3 Vector for Gene Therapy of Hemophilia B.

Although recombinant adeno-associated virus serotype 8 (AAV8) and serotype 5 (AAV5) vectors have shown efficacy in Phase 1 clinical trials for gene therapy of hemophilia B, it has become increasingly clear that these serotypes are not optimal for transducing primary human hepatocytes. We have previously reported that among the 10 most commonly used AAV serotypes, AAV serotype 3 (AAV3) vectors are the most efficient in transducing primary human hepatocytes in vitro as well as in "humanized" mice in vivo, and suggested that AAV3 vectors expressing human coagulation factor IX (hFIX) may be a more efficient alternative for clinical gene therapy of hemophilia B. In the present study, we extended these findings to develop an AAV3 vector incorporating a compact yet powerful liver-directed promoter as well as optimized hFIX cDNA sequence inserted between two AAV3 inverted terminal repeats. When packaged into an AAV3 capsid, this vector yields therapeutic levels of hFIX in hemophilia B and in "humanized" mice in vivo. Together, these studies have resulted in an AAV3 vector predicted to achieve clinical efficacy at reduced vector doses, without the need for immune-suppression, for clinical gene therapy of hemophilia B.

Using Variance to Explore the Diagnostic Utility of Baseline Concussion Testing.

The Graded Symptom Checklist (GSC), Standardized Assessment of Concussion (SAC), Balance Error Scoring System (BESS), and King-Devick Test (KDT) are considered important components of concussion assessment. Whether baseline testing improves the diagnostic utility of these tests remains unclear. We performed an observational cohort study to investigate the within-subject and between-subjects variability of these tests over repeated assessments during two football seasons to examine whether baseline testing reduces variability in test performance. Thirty-five male collegiate football players completed weekly clinical concussion assessments over two seasons. Within-subject (week-to-week) and between-subjects (player-to-player) variability for each test were compared using a bootstrap analysis. Within-subject and between-subjects proportions of overall variance for each test score were calculated. Mixed-model analyses were used to quantify practice effects resulting from repeated testing. For the GSC and BESS, within-subject and between-subjects variability did not significantly differ. For the KDT, the proportion of within-subject variance (20.2%) was significantly less than the between-subjects variance (79.8%). For SAC, however, the proportion of within-subject variance (66.8%) was significantly greater than the between-subjects variance (33.8%). A small, but significant, practice effect was observed for the BESS and KDT tests. When athletes are evaluated during a football season for concussion using the GSC, SAC, and BESS, comparing their scores to baseline performance is likely no more beneficial than comparing them to normative population data for identifying neurological changes associated with concussion. For the KDT, comparison to baseline testing is likely beneficial because of significantly higher between-subjects variability.

Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A.

Potency is a key optimization parameter for hemophilia A gene therapy product candidates. Optimization strategies include promoter engineering to increase transcription, codon optimization of mRNA to improve translation, and amino-acid substitution to promote secretion. Herein, we describe both rational and empirical design approaches to the development of a minimally sized, highly potent AAV-fVIII vector that incorporates three unique elements: a liver-directed 146-nt transcription regulatory module, a target-cell-specific codon optimization algorithm, and a high-expression bioengineered fVIII variant. The minimal synthetic promoter allows for the smallest AAV-fVIII vector genome known at 4,832 nt, while the tissue-directed codon optimization strategy facilitates increased fVIII transgene product expression in target cell types, e.g., hepatocytes, over traditional genome-level codon optimization strategies. As a tertiary approach, we incorporated ancient and orthologous fVIII sequence elements previously shown to facilitate improved biosynthesis through post-translational mechanisms. Together, these technologies contribute to an AAV-fVIII vector that confers sustained, curative levels of fVIII at a minimal dose in hemophilia A mice. Moreover, the first two technologies should be generalizable to all liver-directed gene therapy vector designs.

Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction.

Optimization of a protein's pharmaceutical properties is usually carried out by rational design and/or directed evolution. Here we test an alternative approach based on ancestral sequence reconstruction. Using available genomic sequence data on coagulation factor VIII and predictive models of molecular evolution, we engineer protein variants with improved activity, stability, and biosynthesis potential and reduced inhibition by anti-drug antibodies. In principle, this approach can be applied to any protein drug based on a conserved gene sequence.

Effects of FVIII immunity on hepatocyte and hematopoietic stem cell-directed gene therapy of murine hemophilia A.

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV.

Motor units in the human medial gastrocnemius muscle are not spatially localized or functionally grouped.

KEY POINTS: Human medial gastrocnemius (MG) motor units (MUs) are thought to occupy small muscle territories or regions, with low-threshold units preferentially located distally. We used intramuscular recordings to measure the territory of muscle fibres from MG MUs and determine whether these MUs are grouped by recruitment threshold or joint action (ankle plantar flexion and knee flexion). The territory of MUs from the MG muscle varied from somewhat localized to highly distributed, with approximately half the MUs spanning at least half the length and width of the muscle. There was also no evidence of regional muscle activity based on MU recruitment thresholds or joint action. The CNS does not have the means to selectively activate regions of the MG muscle based on task requirements. ABSTRACT: Human medial gastrocnemius (MG) motor units (MUs) are thought to occupy small muscle territories, with low-threshold units preferentially located distally. In this study, subjects (n = 8) performed ramped and sustained isometric contractions (ankle plantar flexion and knee flexion; range: ∼1-40% maximal voluntary contraction) and we measured MU territory size with spike-triggered averages from fine-wire electrodes inserted along the length (seven electrodes) or across the width (five electrodes) of the MG muscle. Of 69 MUs identified along the length of the muscle, 32 spanned at least half the muscle length (≥ 6.9 cm), 11 of which spanned all recording sites (13.6-17.9 cm). Distal fibres had smaller pennation angles (P < 0.05), which were accompanied by larger territories in MUs with fibres located distally (P < 0.05). There was no distal-to-proximal pattern of muscle activation in ramp contraction (P = 0.93). Of 36 MUs identified across the width of the muscle, 24 spanned at least half the muscle width (≥ 4.0 cm), 13 of which spanned all recording sites (8.0-10.8 cm). MUs were not localized (length or width) based on recruitment threshold or contraction type, nor was there a relationship between MU territory size and recruitment threshold (Spearman's rho = -0.20 and 0.13, P > 0.18). MUs in the human MG have larger territories than previously reported and are not localized based on recruitment threshold or joint action. This indicates that the CNS does not have the means to selectively activate regions of the MG muscle based on task requirements.

Loud preimpact tones reduce the cervical multifidus muscle response during rear-end collisions: a potential method for reducing whiplash injuries.

BACKGROUND CONTEXT: Neck muscle responses after unexpected rear-end collisions consist of a stereotypical combination of postural and startle responses. Prior work using surface electromyography (EMG) has shown that the superficial neck muscle responses can be attenuated when a loud tone (105 dB) is presented 250 milliseconds before impact, but the accompanying response of the deeper multifidus muscles remains unknown. Quantifying this response in multifidus is important because this muscle attaches directly to the cervical facet capsule and can potentially increase the strain in the capsule during an impact and contribute to whiplash injury. PURPOSE: To investigate if a loud preimpact tone decreases the cervical multifidus muscle response during rear-end perturbations. STUDY DESIGN: After approval by the University Clinical Ethics Review Board, human volunteers experienced a series of three whiplash-like perturbations. PATIENT SAMPLE: Twelve subjects with no history of neurologic disorders or whiplash injury were recruited to participate in this experiment. OUTCOME MEASURES: Bilateral indwelling EMG of multifidus at the C4 and C6 levels, surface EMG of sternocleidomastoid (SCM) and C4 paraspinals (PARAs), and kinematics of the head/neck were measured. METHODS: Subjects experienced three whiplash-like perturbations (peak acceleration of 19.5 m/s(2)) preceded by either no tone or a loud tone (105 dB) presented 250 milliseconds before sled acceleration onset. RESULTS: The loud tone decreased the muscle activity of C6 multifidus (42%) and C4 PARAs (30%), but did not affect the C4 multifidus or SCM activity. Peak head kinematic responses (extension angle: 6%, retraction: 9%, linear forward acceleration: 9%, and angular acceleration in extension: 13%) were also decreased by the loud preimpact tone. CONCLUSIONS: The attenuation of peak C6 multifidus activity and head kinematic responses suggests that a loud preimpact tone may reduce the strain in the cervical facet capsule, which may reduce the risk of whiplash injury during rear-end collisions.

Development and validation of an objective balance error scoring system.

INTRODUCTION: Limited access to sophisticated technology and the unreliability of simple tools prevent accurate and reliable human standing balance assessments outside research laboratory settings. The goal of this study was to develop and validate a simple objective balance assessment tool that provides an accurate, reliable, and affordable alternative to currently available laboratory and clinical methods. METHODS: Thirty healthy subjects were filmed performing the Balance Error Scoring System (BESS) while wearing inertial measurement units (IMU) measuring linear accelerations and angular velocities from seven locations of the body: forehead, sternum, waist, right and left wrist, and right and left shin. Each video was scored by four experienced BESS raters, whose mean scores were used to develop an algorithm computing objective BESS (oBESS) scores solely from IMU data. Interrater reliability and accuracy of oBESS scores were assessed using intraclass correlations (ICC). RESULTS: Raters displayed low variability in scoring (ICC3,1 = 0.91). The oBESS was able to produce scores with accurate fit to raters (ICC3,1 = 0.92) and predicted individual BESS scores (ICC3,1 = 0.90) using data from one IMU placed at the forehead. oBESS was unable to produce accurate scores (ICC3,1 = 0.68) when using IMU data from the subset of conditions (firm surface only) used in popular concussion identification protocols. CONCLUSION: The oBESS can reliably predict total BESS scores in healthy subjects. Pending further validation, oBESS could represent a valid tool to assess balance by offering an objective and reliable alternative to the current scoring methods of the BESS.

Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

Five-year experience with donation after cardiac death kidney transplantation in a Canadian transplant program: Factors affecting outcomes.

BACKGROUND: : Donation after cardiac death (DCD) has led to an increase of up to 40% in the number of kidney transplants in some programs. Unfortunately, the increase in warm ischemic time results in higher rates of delayed graft function (DGF). The purpose of our study was to examine our initial 5-year experience with DCD kidney transplantation and to determine the factors involved in early postoperative function and function at 1 year. METHODS: : This retrospective study included a review of the recipient and donor charts of 63 DCD kidneys retrieved and transplanted by the London Multi-Organ Transplant Program between July 2006 and October 2011. Comparisons were carried out between our early (n=31, July 2006 to January 2009) and our recent experience (n=32, March 2009 to October 2011). DGF and creatinine clearance at 3, 7 and 365 days were examined with regression analyses. RESULTS: : DGF was seen in 65% of transplanted kidneys. Mean creatinine clearance (CrCl) at 1 year was 66.7 mL/min. Low pre-transplant recipient daily urine output was the most statistically significant predictor of DGF in multivariate analysis (p < 0.001). In comparisons between our early and more recent results, improvements were noted in time from asystole to flush (16.0 vs. 12.0 minutes, p = 0.003), while cold ischemic time increased (464 vs. 725 minutes, p = 0.006). Experience contributed to a significant reduction in hospital length of stay (16 vs. 13 days, p = 0.035) and improved early renal function (CrCl at 3 days 7.8 vs. 11.9 mL/min, p = 0.027). The use of machine cold perfusion and higher recipient preoperative daily urine output predicted improved early renal function, while increasing donor age predicted poorer function at 1 year. DISCUSSION: : Despite early DGF, our results justify the continued transplantation of kidneys from DCD donors.

Enhanced biosynthesis of coagulation factor VIII through diminished engagement of the unfolded protein response.

Human and porcine coagulation factor VIII (fVIII) display a biosynthetic efficiency differential that is being exploited for the development of new protein and gene transfer-based therapies for hemophilia A. The cellular and/or molecular mechanism(s) responsible for this phenomenon have yet to be uncovered, although it has been temporally localized to post-translational biosynthetic steps. The unfolded protein response (UPR) is a cellular adaptation to structurally distinct (e.g. misfolded) or excess protein in the endoplasmic reticulum and is known to be induced by heterologous expression of recombinant human fVIII. Therefore, it is plausible that the biosynthetic differential between human and porcine fVIII results from differential UPR activation. In the current study, UPR induction was examined in the context of ongoing fVIII expression. UPR activation was greater during human fVIII expression when compared with porcine fVIII expression as determined by ER response element (ERSE)-luciferase reporter activity, X-box-binding protein 1 (XBP1) splicing, and immunoglobulin-binding protein (BiP) up-regulation. Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII was notably absent in the Golgi apparatus, confirming that endoplasmic reticulum to Golgi transport is rate-limiting. In contrast, a significant proportion of porcine fVIII was localized to the Golgi indicating efficient transit through the secretory pathway. Overexpression of BiP, an integral UPR protein, reduced the secretion of human fVIII by 50%, but had no effect on porcine fVIII biosynthesis. In contrast, expression of BiP shRNA increased human fVIII expression levels. The current data support the model of differential engagement of UPR by human and porcine fVIII as a non-traditional mechanism for regulation of gene product biosynthesis.

Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein.

A point mutation leading to amino acid substitution N1922S in the A3 domain of factor VIII (fVIII) results in moderate to severe hemophilia A. A heterologous expression system comparing N1922S-fVIII and wild-type fVIII (wt-fVIII) demonstrated similar specific coagulant activities but poor secretion of N1922S-fVIII. Immunocytochemical analysis revealed that intracellular levels of N1922S-fVIII were similar to those of wt-fVIII. The specific activity of intracellular N1922S-fVIII was 10% of that of wt-fVIII, indicating the presence of large amounts of a nonfunctional N1922S-fVIII-folding intermediate. wt-fVIII colocalized with both endoplasmic reticulum (ER)- and Golgi-resident proteins. In contrast, N1922S-fVIII colocalized only with ER-resident proteins, indicating a block in transit from the ER to the Golgi. A panel of conformation-dependent monoclonal antibodies was used to determine native or nonnative folding of N1922S-fVIII. Intracellular N1922S-fVIII but not secreted N1922S-fVIII displayed abnormal folding in the A3 and C1 domains, indicating that the A1, A2, and C2 domains fold independently into antigenically intact tertiary structures, but that folding is stalled in the mutant A3 and its contiguous C1 domain. In summary, the N1922S substitution results in poor secretion of a functional protein, and the domain-specific defect in folding and intracellular trafficking of N1922S-fVIII is a novel mechanism for secretion defects leading to hemophilia A.