Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction

Zakas, Philip M; Brown, Harrison C.; Knight, Kristopher A.; Meeks, Shannon L.; Spencer, H. Trent; Gaucher, Eric A.; Doering, Christopher B.

doi:10.1038/nbt.3677

Cited by 89 publications

(80 citation statements)

References 36 publications

(59 reference statements)

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“…In general, ancestral enzymes are considered to be generalists having a broader range of applicability than contemporary enzymes, which are considered specialists 37 , including extremophiles, for which the evolution to substrate and organismal specificity may limit their efficiency outside their natural environment. Thus, ASR emerges as a potential methodology for protein engineering with multiple applications in biotechnology 38,39 , beyond its possible evolutionary implications. Also, it is remarkable that our resurrected EG enzyme works well at 30°C.…”

Section: Discussionmentioning

confidence: 99%

Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

et al. 2019

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Cellulases catalyze the hydrolysis of cellulose. Improving their catalytic efficiency is a longstanding goal in biotechnology given the interest in lignocellulosic biomass decomposition. Although methods based on sequence alteration exist, improving cellulases is still a challenge. Here we show that Ancestral Sequence Reconstruction can "resurrect" efficient cellulases. This technique reconstructs enzymes from extinct organisms that lived in the harsh environments of ancient Earth. We obtain ancestral bacterial endoglucanases from the late Archean eon that efficiently work in a broad range of temperatures (30-90°C), pH values (4-10). The oldest enzyme (~2800 million years) processes different lignocellulosic substrates, showing processive activity and doubling the activity of modern enzymes in some conditions. We solve its crystal structure to 1.45 Å which, together with molecular dynamics simulations, uncovers key features underlying its activity. This ancestral endoglucanase shows good synergy in combination with other lignocellulosic enzymes as well as when integrated into a bacterial cellulosome.

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Section: Discussionmentioning

confidence: 99%

Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

et al. 2019

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“…Despite the treatment of over 100 E16 hemophilia A mice on a mixed S129-C57BL/6 background with varying doses of AAV-HCB-fVIII vectors, no fVIII inhibitor development has been observed. This finding is independent of the fVIII transgene used as BDD-hfVIII, ET3, and ancestral fVIII variants (e.g., An53) all have been tested, and despite the presence of up to 10% non-human sequence, no antibody formation has been observed [unpublished data as well as (23,86)]. In contrast, other groups clearly demonstrated inhibitor development using codon-optimized BDD-hfVIII transgenes driven by alternative synthetic promoters.…”

Section: Murine Preclinical Studiesmentioning

confidence: 93%

“…Over the past decades, several studies have demonstrated preclinical efficacy following liver-directed AAV-fVIII gene therapy (21)(22)(23)(81)(82)(83)(84)(85)(86)(87). However, these studies vary greatly in respect to pharmacological parameters including study duration, vector doses, fVIII transgene design, experimental species utilized, and/or the use of immunodeficient animals or transient immune suppression to obviate immune complications ( Table 1).…”

Section: Murine Preclinical Studiesmentioning

confidence: 99%

The Immune Response to the fVIII Gene Therapy in Preclinical Models

et al. 2020

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Neutralizing antibodies to factor VIII (fVIII), referred to as "inhibitors," remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product-and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liverspecific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pretransplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, Patel et al. Preclinical Gene Therapy fVIII Immunology respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.

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“…The analysis of these differences can lead to insights towards leveraging bioengineered forms with enhanced functional properties. Zakas et al have gone beyond the work based on ortholog scanning that led to the ET3 FVIII construct and have generated higher resolution mapping of FVIII protein sequences through comparisons of sequential phylogeny branches. This led to the identification of ancestral FVIII constructs which demonstrated significantly higher FVIII production compared to human FVIII.…”

Section: Bioengineering Of Fviiimentioning

confidence: 99%

Bioengineered molecules for the management of haemophilia: Promise and remaining challenges

Pipe

2018

Haemophilia

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Recombinant DNA technology has led to accelerating introduction of novel therapeutics for the treatment of haemophilia. This technology has driven the development of recombinant clotting factors, extended half-life clotting factors, alternative biologics to promote haemostasis and enabled the launch of the gene therapy era for haemophilia. At the core of this technology is the ability to study the structure and function of the native molecules and to apply rational bioengineering to overcome limitations to the existing therapies. Through the study of haemophilia-causing mutations, site-directed mutagenesis, detailed structural models and a wide repertoire of animal models, new bioengineering strategies are helping overcome some of the remaining limitations and challenges of traditional clotting factor concentrates. Some of these bioengineering strategies are now being partnered with improvements in vectorology leading to the first wave of successful gene therapy approaches. This study will review past and present bioengineered molecules that are advancing care for haemophilia as well as novel approaches that promise to continue to improve care and outcomes for patients with haemophilia.

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Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction

Cited by 89 publications

References 36 publications

Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

The Immune Response to the fVIII Gene Therapy in Preclinical Models

Bioengineered molecules for the management of haemophilia: Promise and remaining challenges

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