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.

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.

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.

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.

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.

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.

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.