Many currently approved gene therapies use adeno-associated virus (AAV) to deliver DNA sequences encoding protein(s)-of-interest into cells. The AAV viral genome forms stable, circular DNA structures called episomes after entering the nuclei. Therapeutic proteins are then generated invivo from transcription and translation of these episomes, and long-term durability thus depends on episome stability. Prior modeling work has utilized differential equation-based models to characterize AAV uptake and subsequent protein production. However, episome loss associated with target cell turnover is poorly described with these models. Here, an agent-based model (ABM) to overcome this shortcoming is developed. The liver was used as the example organ as it is known to be self-renewing and has been a common target for gene therapies. In this model, each hepatocyte is an agent, capable of division and death. When transduced, these agents acquire and carry episomes. During cell division, episomes are passed from mother to daughter cells. All episomes are presumed lost when transduced cells die. The ABM was applied to etranacogene dezaparvovec (formerly AMT-061 or CSL222), a liver-targeting gene therapy for hemophilia B consisting of AAV serotype 5 particles encoding a transgene for the Padua variant (R338L) of coagulation factor IX (FIX). ABM-simulated FIX activity in patients receiving this therapy was consistent with clinical observations over more than 2 years following treatment. This ABM was then used to generate 20-year predictions and explore biological mechanisms underlying long-term durability. The ABM framework and approach should be applicable to investigating durability of other AAV-based gene therapies.

Consensus computational immunogenicity modelling of botulinum neurotoxin serotypes: Cross-platform validation, uncertainty quantification, and relative risk assessment.

Ion channels are central to regulating neuronal communication, cardiac rhythm, and muscle contraction. Their modulation can induce therapeutic benefits but may also lead to adverse or toxic effects. This study presents IonNTxPred, a protein language model (PLM)-based method for predicting protein ion channel modulators, including channel-specific (such as sodium, potassium, calcium, and others) and moonlighting proteins capable of modulating multiple ion channels. We train, test, and evaluate our models on the largest dataset of non-redundant ion channel modulating proteins, where no two proteins have more than 40% sequence identity. Composition analysis revealed that residues Cys, Gly, and Trp are highly prevalent, whereas Ala, Glu, Leu, Gln, and Val are scarce in ion channel modulating proteins, with Cys identified as a key discriminative residue. We explored both alignment-based (BLAST, MERCI) and alignment-free (machine learning, deep learning, and PLM-based) approaches. Among these, our evolutionary information-based PLM (ESM2-t33) achieved the best performance, with an AUROC of 0.97 across ion channels, which further improved to 0.98 when integrated with BLAST output. The proposed method outperformed existing approaches on independent datasets. We used IonNTxPred to screen FDA-approved and organismal proteins to identify candidates with ion channel modulating potential, supporting drug repurposing, discovery of new therapeutic proteins, and safety assessment of existing biologics. We implemented these models in a user-friendly web server, IonNTxPred, which facilitates the design and discovery of ion channel modulating protein-based drugs and supports the biosafety evaluation of therapeutic proteins through neurotoxin screening (https://webs.iiitd.edu.in/raghava/ionntxpred/).

Biopharmaceuticals, such as monoclonal antibodies (mAb), are complex molecules that require thorough analysis due to post-translational modifications, including charge variants and glycoform patterns. When analyzing intact mAbs, single liquid chromatography (LC) mass spectrometry (MS) methods often lack the resolution to distinguish this heterogeneity because they offer limited separation in both chromatographic and mass dimensions. Consequently, these methods struggle to detect low-abundance species, highlighting the importance of 2DLC-MS workflows. Here, we demonstrate the potential of the offline ion exchange chromatography (IEC)-hydrophilic interaction chromatography (HILIC)-MS method to effectively separate charge and glycoform variants of complex mAbs. Cetuximab served as the model antibody due to its multiple charge variants (arising from sialylation and C-terminal lysine clipping) and four glycosylation sites (in both Fab and Fc regions). Charge variants were first separated using non-volatile IEC and collected as individual fractions. These fractions were then analyzed using a low-flow HILIC-MS setup to increase sensitivity for low-abundance species. A RPLC trap columnwas used to desalt and reduce the volume transferred tothe HILICseparation. The IEC-HILIC results showed that both Fc- and Fab-associated glycoforms of cetuximab were resolved to a level not achievable with HILIC-MS alone, allowing deeper insights into charge heterogeneity and glycosylation. In addition to resolving combined Fab/Fc glycoforms, the method also distinguished Fc-only glycoforms from the main isoform. This orthogonal IEC-HILIC method provides additional insights into charge and glycosylation heterogeneity, enabling a more comprehensive analysis of cetuximab. It provides an effective approach for detailed characterization of charge variations and glycoform diversity in next-generation protein therapeutics.

THPC modification converts cytochrome c into a self-delivering anticancer protein. The conjugate escapes endosomal degradation in a manner dependent on endolysosomal acidification, activates caspase-9/3, and induces apoptosis in cancer cells. This work offers a simple, carrier-free strategy for cytosolic protein therapeutics.

Antibodies and other protein therapeutics have revolutionized medicine, but their application is largely limited to extracellular targets. The lack of efficient intracellular delivery methods remains a major bottleneck. Here, we engineered a family of small (∼90 amino acids), metabolically stable membrane translocation domains (MTDs) by modifying the loop sequences of a human fibronectin type III (FN3) domain. The most potent variant, MTD4, is highly cell-permeable and can be recombinantly fused to the N- or C-terminus of any peptide or protein, serving as a versatile delivery vehicle. We demonstrate that MTD4 fusions efficiently deliver a wide variety of functional peptides and proteins into the cytosol and nucleus of eukaryotic cells, both in vitro and in vivo. Following systemic administration, an MTD4 fusion protein exhibited broad biodistribution and homogeneous tissue penetration in mice. Importantly, MTD4 is effective at low nanomolar (nM) concentrations, making it a promising platform for addressing a vast range of intracellular and previously ″undruggable″ targets.

Artificial intelligence (AI) is reshaping drug discovery by bridging the gap between traditional computer-aided drug design (CADD) and next-generation, data-driven methodologies. Unlike conventional CADD, which relies on physical modelling of molecular interactions, AI integrates machine learning (ML) and deep learning (DL) to leverage rapidly expanding datasets in biology and chemistry. These approaches enable efficient prediction of molecular structures, binding affinities, and pharmacological properties, thereby reducing both time and cost in drug development. The impact is particularly profound in the design of protein therapeutics, such as antibodies, which require accurate modelling of complex structures and interactions. Emerging AI frameworks, including generative adversarial networks (GANs), reinforcement learning (RL), and multi-omics integration, are accelerating target identification, optimizing lead candidates, and refining pharmacokinetic and biophysical profiles. In this review, we highlight recent advances at the interface of AI and antibody drug discovery, discuss key methodological developments, and examine the challenges that remain in translating AI-driven strategies into clinical success. We further explore how AI-enabled platforms are redefining the landscape of precision biopharmaceuticals, offering new opportunities for efficient and targeted therapeutic development.

Therapeutic peptides and proteins form higher-order structures (HOS) including oligomeric forms in formulation, critical for drug efficacy, safety, and stability. To quickly assess protein oligomerization, dynamic light scattering (DLS) was applied to measure a series of protein standards with molecular weight (MW) range of 1.3-660kDa, yielding translational diffusion coefficients (Ddls), which were corrected to Dcorr using water diffusion data. A correlation between Dcorr and protein monomeric MW, representing their hydrodynamic MW (MWhd), was established as . This DLS/MWhd model was subsequently applied to therapeutic protein formulations with monomeric MW ranging from 3.8 to 149kDa. The resulting MWhd values were several-fold greater than the corresponding monomeric MW of the glucagon-like peptide-1/2 (GLP-1/2) analogs, insulin analogs, and the monoclonal antibodies (mAbs) infliximab and bevacizumab, indicating varying degrees of oligomerization. The observed oligomerization states were largely consistent with those reported in literature. For insulin and mAb oligomers, pseudo-spherical diffusion coefficients (Ds) back calculated from the oligomer MW agreed within 6% of experimental Dcorr values, except for the insulin dimer. Incorporation of Perrin's anisotropic correction reduced insulin dimer discrepancy from 9 to 5%. Overall, the established MWhd model, incorporating a larger exponent of 0.428 that accounts for protein anisotropy, provides a rapid, non-invasive method to factually assess protein oligomerization in as-is formulations, aiding drug development and quality control. Minor discrepancies relative to size exclusion chromatograph (SEC) or prior biophysical studies may arise from experimental differences in protein concentration, oligomeric equilibrium, pH, excipients or modeling assumptions, warranting further investigation.

Lipid nanoparticles (LNPs) containing mRNA that encodes therapeutic proteins have emerged as apromising therapeutic modality for a myriad of diseases. However, the pharmacokinetic (PK) relationships between the nanoparticle carrier, its mRNA payload, and the expressed protein remain poorly understood. Here we have investigated whole-body PK of these components in mice following intravenous administration of an mRNA-LNP that expresses a non-cross-reactive monoclonal antibody. LNPs encapsulating mRNA were prepared via microfluidic mixing and characterized for physicochemical properties and encapsulation efficiency. Following a single intravenous administration of mRNA-LNP, blood, plasma, and tissues were collected for PK measurement over 2weeks. Ionizable lipid (i.e., ALC-0315) concentrations were determined using LC-MS, mRNA concentrations were measured using qPCR, and the expressed antibody concentrations were determined using ELISA. It was found that each component exhibited a distinct PK profile. For example, lipid exposure was highest in liver and spleen, while mRNA accumulation peaked in spleen and heart. Strikingly, the expressed antibody demonstrated a distribution pattern that did not mirror mRNA exposure, with the lung showing the greatest antibody levels despite only modest mRNA delivery. Tissue-to-plasma ratios for the expressed antibody exceeded values reported for intravenously administered antibodies, suggesting localized production or enhanced retention following in situ translation. These findings highlight that functional protein exposure is governed by tissue-specific translational efficiency and protein properties, rather than just nanoparticle delivery. The comprehensive PK data presented here also provides afoundation for the development of systems-based PK models for antibody expressing mRNA-LNPs.

The multi-attribute method (MAM) is an integrated peptide mapping strategy based on liquid chromatography-mass spectrometry (LC-MS) technology. It enables precise quantification and dynamic tracking of multiple site-specific modifications in a single analysis, significantly enhancing the efficiency and depth of biopharmaceutical quality control. Notably, its integrated application across process development, process monitoring, and product release has driven a paradigm shift from a "single-attribute, single-method" approach to a "multi-attribute, integrated-method" approach in quality control. This review systematically summarizes the technical principles, optimization strategies, and application progress of MAM by integrating recent research cases of complex therapeutic proteins (e.g., monoclonal antibodies and Fc fusion proteins), with a focus on specific strategies and practical paths of MAM in workflow automation, new peak detection (NPD) optimization, intact multi-attribute method (iMAM), and the integration of complementary technologies. The objective is to provide a valuable reference for the standardization and industrial application of MAM in biotechnological drug quality control. Although MAM is expected to become a core analytical tool for biopharmaceutical quality control, its widespread industrial application remains constrained by key challenges, including insufficient method robustness, incomplete standardization, and variable regulatory acceptance. Notably, a significant stride in regulatory acceptance has been made with the recent implementation of the United States Pharmacopeia (USP) General Chapter < 1060 > , which establishes the first official framework for MAM. Beyond this regulatory milestone, future efforts should focus on advancing automated platform development, creating intelligent data algorithms, and strengthening cross-disciplinary collaboration to further promote the systematic integration and standardized application of MAM throughout the full lifecycle quality management of biotechnological drugs.

Chinese hamster ovary (CHO) cells are the leading host for recombinant therapeutic protein production in the biopharma industry. In this study, we investigated how feeding acidic forms of tricarboxylic acid (TCA) cycle intermediates-malic acid, succinic acid, and α-ketoglutaric acid-affects cell culture performance and metabolism in two industrial IgG-producing CHO cell lines. These intermediates were used as pH control agents to replace conventional CO2 sparging, enabling simultaneous modulation of the bioreactor environment and cell metabolism. Carbon-13 metabolic flux analysis (13C-MFA) revealed substantial rewiring of central carbon and nitrogen metabolism under all fed conditions, with distinct responses between cell lines. Intermediate-fed cultures exhibited enhanced TCA cycle fluxes, reduced glucose dependency, decreased lactate accumulation, and altered routing of pyruvate. Notably, α-ketoglutaric (α-KG) acid feeding triggered divergent nitrogen assimilation phenotypes: one cell line enhanced glutamine biosynthesis and ammonium clearance, while the other accumulated glutamate with minimal glutamine production. These metabolic adaptations were accompanied by shifts in redox balance and delayed but measurable increases in cell-specific IgG productivity. Our findings highlight the compound- and cell line-specific nature of metabolic responses to TCA cycle intermediate feeding and support its use for pH control and bioprocess optimization.

ABSTRACT Recombinant proteins with applications in human and veterinary medicine are mainly produced in mammalian expression systems, particularly Chinese hamster ovary (CHO) cells. Although these cells enable proper folding and post-translational modifications, they often exhibit low specific productivity, which represents a critical limitation in large-scale biopharmaceutical manufacturing. Strong viral promoters, including CMV and its enhancer-enriched variant CMV+E, are commonly used to drive transgene expression. However, they are susceptible to epigenetic silencing and progressive loss of activity during prolonged culture, which can compromise yield stability. Endogenous host-cell promoters constitute a promising alternative, as they can maintain transgene expression in alignment with physiological and bioprocess regulatory mechanisms. Here, we assessed the activity of endogenous eukaryotic promoters previously identified in our laboratory from highly expressed CHO-K1 genes. The receptor-binding domain (RBD) of SARS-CoV-2 was selected as a model of a complex secretory glycoprotein domain. Our results demonstrate that the Hspa5 and Vim promoters enable sustained and high-level expression of recombinant RBD protein in CHO-K1 cells, achieving performance comparable to the enhancer-containing viral promoter CMV+E. Notably, Hspa5 and Vim are unoptimized genomic sequences lacking classical enhancers; nevertheless, their intrinsic stability and reduced susceptibility to silencing make them attractive regulatory elements for recombinant protein production. These findings highlight their potential for next-generation promoter engineering and for the development of long-term, high-yield mammalian expression systems for complex therapeutic proteins.

Neutralizing antibody (NAb) activity assessment is a regulatory expectation in the clinical development of therapeutic proteins, traditionally addressed through standalone NAb assays. However, evolving regulatory guidance and industry experience increasingly recognize that integrated analyses of anti-drug antibodies (ADA), pharmacokinetics (PK), pharmacodynamics (PD), efficacy, and safety may provide a more clinically meaningful evaluation of neutralizing activity. This perspective examines the scientific and regulatory rationale for moving beyond routine reliance on standalone NAb assays and describes a risk-based, integrated immunogenicity assessment framework. Two case studies involving low-risk monoclonal antibodies-garadacimab and clazakizumab-are presented, in which validated standalone NAb assays were available but not relied upon for primary clinical interpretation due to limited sensitivity and lack of added clinical value. Instead, longitudinal ADA characterization integrated with PK and PD or efficacy data was used to assess clinically meaningful neutralizing activity. In both cases, this approach enabled clear differentiation between detectable but clinically irrelevant immunogenicity and immunogenicity with clinical consequence and was accepted by regulatory authorities. These examples illustrate how integrated immunogenicity assessments can replace standalone NAb assays while remaining scientifically rigorous, clinically informative, and aligned with regulatory expectations.

Currently, asymmetric polymeric membranes exhibit numerous advantages for wound dressing applications. Usually, numerous systems allow the release of only one or several antibacterial drugs to fight against the bacteria present on the wound. However, to be efficient, it is necessary to disrupt biofilm formation in order to render the enclosed bacteria sensitive to the antibacterial agent. Here, we describe a system capable of achieving such goal via a double compartment asymmetric polymeric membrane designed for dual drug release. In view of elaborating our gradient-structured membrane for differential kinetic release of combined antibiofilm/antibiotic agents in the context of wound dressing applications, a previously electrospun poly(vinyl alcohol) (PVA) fibrous membrane (EFM) was combined with a renewable poly(butylene-succinate-co-adipate) (PBSA) asymmetric porous membrane (AM) via physical adhesion. Physical adhesion was promoted via surface modification of the PVA fibers involving complexation of the numerous PVA hydroxyl groups with phenyl boronic acid (PBA). This modification resulted in heightened hydrophobicity of the upper EFM layer with substantial contact angle increase up to 115° allowing for effective adhesion of the PVA based EFM onto the AM. No delamination was observed. Thus, thanks to surface modification, a gradient-structured double compartment asymmetric membrane (DCAM) was obtained consisting of a dense/macro-/micro-/nano-porous structure. Furthermore, as proof of concept, our study shows that PBA can be used as a pro-drug mimic pH sensitive system exhibiting release profiles in aqueous environment up to 3-fold higher in acidic compared to that for neutral or alkaline environments. Moreover, the BSA (Bovine Serum Albumin) use as a potential therapeutic model protein was encapsulated within the porous structure and its release profile was monitored over time showing maximum release attained within 24 h. Finally, the biocompatibility of our new DCAM was confirmed via the internationally recognized standard ISO 10993-5: 2009 assay for the in vitro cytotoxicity testing of medical devices. Our results are promising in that they provide a new structural substrate for the dual concomitant differential-controlled release of large amounts of high (antibiofilm and potentially other therapeutic proteins) and low molecular weight (antibacterial) bioactive agents.

Abstract Pediatric high-grade glioma (pHGG) have a poor prognosis with limited therapeutic options. Cellular therapies, like CAR-T therapy, have revolutionized the treatment of some cancers. Unfortunately, T-cell based therapies have so far shown limited efficacy for brain tumors, due to the lack of unique tumor antigens, the downregulation of targeted antigens, and the immune suppressive tumor microenvironment (TME). To circumvent these obstacles, we developed a cellular delivery system where implanted post-mitotic Migratory Inhibitory Interneuron Precursors (MIPs) migrate to pHGG by chemoattraction and induce a cytotoxic tumor response via secretion of bispecific T-cell engagers (BiTEs). The inhibitory interneurons of the cerebral cortex originate predominantly in the ventral/subcortical portion of the telencephalic neural tube. During fetal brain development, canonical chemoattractants induce MIP migration over long distances from subcortical origins to the cerebral cortex. MIPs can be differentiated from ES cells or iPSCs. Our in vitro and in vivo data show that MIPs robustly migrate to the majority of pHGG evaluated. We have also modified these MIPs to drive chemoattraction to other factors in order to expand the number of pHGG MIPs can migrate to. As a proof of principle, we were able to significantly prolong (double) surival of pHGG mouse models by injecting MIPs that locally, at the tumor, secrete an EGFR-BiTE that engages with bystander T-cells. It is our goal for our therapy to become fully T-cell independent, therefore we are exploring the delivery of alternate therapeutic proteins.

Rational design of PEGylated protein therapeutics requires mechanistic understanding of how coupling chemistry and PEG size jointly determine product homogeneity, receptor engagement, and structural integrity─the properties that collectively govern whether a conjugate is a viable candidate for further development. Anakinra (recombinant interleukin-1 receptor antagonist, IL-1Ra) is an approved anti-inflammatory biologic whose short systemic residence necessitates daily subcutaneous injection and is associated with steep peak-trough exposure fluctuations that, in chronic high-dose regimens, have been linked to IL-1Ra-derived systemic amyloidosis. PEGylation offers a chemically established route to increase the apparent molecular size of IL-1Ra and thereby reduce renal filtration and smooth exposure, but the structural and functional consequences of conjugation have never been characterized at residue resolution for this target─a gap that limits informed design choices between available chemistries. Here, we address this gap by establishing a head-to-head characterization framework comparing site-specific thiol-maleimide conjugation and random amino-coupling on a uniformly 15N-labeled recombinant IL-1Ra variant (M143V) that preserves all native conjugation sites and is potency-matched to Anakinra. Across a 5-20 kDa PEG ladder at a substoichiometric 0.5:1 PEG:protein feed, site-specific thiol-maleimide coupling yielded substantially higher conversion (∼81%) and a homogeneous single-species di-PEGylated (DoP 2) conjugate, whereas random amino-coupling produced heterogeneous mixtures across all PEG sizes (∼33-36% conversion, multiple degrees of polymerization). Purified single-species conjugates were benchmarked under a fixed IL-1β EC80 challenge in HEK-Blue reporter cells; all retained full maximal efficacy but exhibited chemistry- and size-dependent right-shifts in IC50, indicating steric modulation of receptor engagement rather than loss of function. To directly establish whether PEG attachment perturbs structural integrity, 2D 1H-15N HSQC NMR on the 15N-labeled thiol-PEG10 conjugate demonstrated a preserved global fold with localized chemical-shift perturbations near cysteine-proximal surface regions, confirming site-selective modification and intact binding scaffold. Together, these data establish the conjugation quality, antagonist activity, and structural integrity of the lead conjugate as the prerequisite characterization foundation for its further development and provide a transferable analytical framework for the rational design of PEGylated cytokine therapeutics.

Native chemical ligation (NCL) represents a powerful strategy for the chemical synthesis of tailor-made proteins that are difficult to obtain via biological expression approaches. Early desulfurization methods, particularly VA-044-based desulfurization, have profoundly advanced the field by enabling the post-ligation conversion of cysteine to alanine, thereby expanding the sequence space accessible for protein synthesis. In recent years, a variety of innovative desulfurization technologies have emerged that achieve superior reaction kinetics, broader functional group compatibility or elimination of harmful additives, significantly broadening the scope and practicality of desulfurization strategies. This review summarizes key enabling desulfurization methodologies developed in recent years, including boron reagent-based desulfurization, photochemically induced reactions, iron-catalyzed systems, and ultrasound-driven processes. We highlight their core mechanisms, technical advantages, and substrate tolerability, and showcase representative applications that have expanded the synthetic protein landscape. Finally, we provide an outlook on future directions, encompassing precise mechanistic elucidation, desulfurization-functionalization tandem strategies, and industrial scale-up, which are expected to further drive innovations in protein therapeutics, chemical biology, and novel biomaterials.

Inhaled biologics to treat lung diseases have an extensive history. This is counterbalanced by the limited products that have reached the commercialization stage of development despite the numerous disease states and targets that may be suitable for the delivery of proteins, peptides, or nucleic acid therapeutics. These opportunities are reviewed alongside current and past clinical and preclinical studies that highlight both successful and discontinued programs, providing key lessons for future development. Although there are physical, biological, and technical barriers that must be overcome to successfully deliver these relatively fragile moieties to the lung, there are commercial and clinical reasons why success has not always been realized. This review captures the current landscape of inhaled biologics, and as the field advances, we expect the inhaled route to expand rapidly given its distinct pharmacokinetic advantages for local lung diseases.

Targeted delivery of protein therapeutics remains challenging for translating biologics into effective treatments. Here, we introduce a universal strategy leveraging elevated glycolysis, a hallmark of many pathological states, and its resulting extracellular acidification as a navigational cue. Therapeutic proteins are encapsulated within pH-responsive polymer shells that remain near-neutral at physiological pH but gradually gain positive charge under acidic conditions. This dynamic charge modulation allows nanocapsules to sense pH gradients between healthy and diseased tissues, directing them toward pathological sites. Unlike receptor-mediated targeting that operates over nanometer scales, this receptor-independent approach enables long-range targeting. In vivo models of cancer, chronic inflammation, and acute injury demonstrate selective accumulation of encapsulated proteins at diseased sites, enhancing therapeutic efficacy while reducing systemic toxicity. By transforming a ubiquitous metabolic signature into a directional driving force, this lactate acid gradient-mediated targeting (LaGET) platform offers a previously underexplored paradigm for targeted delivery of protein therapeutics.

In pursuit of a gene transfer agent with efficient pulmonary transduction, the UK Respiratory Gene Therapy Consortium has developed a lentiviral vector pseudotyped with the envelope proteins, F and HN from Sendai virus (rSIV.F/HN). In contrast to other viral vectors, pulmonary rSIV.F/HN delivery achieves sustained gene expression ( ~ 2 years in mice) in the lungs and systemic circulation following a single dose. Here, we investigate the application of the rSIV.F/HN vector-platform for wider indications, including systemic disorders that require serum expression of therapeutic proteins. To assess the potential for rSIV.F/HN to produce systemic proteins, intravenous vector delivery was characterised and compared against intrapulmonary administration, achieved via 'nasal sniffing'. Both delivery routes achieved sustained (at least 1 year) systemic expression of the secreted reporter protein Gaussia luciferase. Systemic rSIV.F/HN delivery resulted in widespread protein expression across multiple organs, accompanied by the generation of significant anti-vector neutralising antibodies limiting vector readministration. Conversely, localised airway transduction was observed following pulmonary administration, which we have previously shown is not an impediment to efficient vector readministration. These data support intrapulmonary rSIV.F/HN delivery for systemic protein production, with sustained high-level transgene expression and feasible readministration.