key: cord-0856463-z8piqvf6
authors: Daniell, Henry
title: From conception to Covid‐19: An arduous journey of tribulations of racism and triumphs
date: 2020-08-16
journal: Plant Biotechnol J
DOI: 10.1111/pbi.13468
sha: 7aab206295f8266e21d1a450a585c5e9099bfb76
doc_id: 856463
cord_uid: z8piqvf6

The densely wooded 800‐acre Madras Christian College (MCC) campus, India where I lived most of my early years, sparked my curiosity in plants. Observing the pitcher plant that captured insects, trees that produced fruits in the shape of a human skull, wild orchids, bright wildflowers, and dense vegetation amidst monsoon rains was exciting but also scary due to the wildlife, especially foxes and snakes. I was born in Salem, India, located at the foothills of mountains, tenth in a family of twelve, where my father was a high court judge.

insects, trees that produced fruits in the shape of a human skull, wild orchids, bright wildflowers, and dense vegetation amidst monsoon rains was exciting but also scary due to the wildlife, especially foxes and snakes. I was born in Salem, India, located at the foothills of mountains, tenth in a family of twelve, where my father was a high court judge. Sadly, he died when I was 12, and my biosynthesis in vitro, at rates higher than in vivo (1980) (1981) (1982) . After completing my post-doctoral training, I returned back to MKU where I developed an in organello system that could synthesize a fully functional photosystem I, macro-grana and photosystem II capable of oxygen evolution. I was able to develop chloroplasts capable of prolonged protein synthesis, for up to eight hours (Daniell et al., 1986a) . Due to unpredictable power failures during the daytime at MKU, I hitched rides in lorries to the university and performed experiments at night, when the power supply was more stable.

During this period, Prof. Bruce McFadden from Washington State University (WSU) visited MKU as a Fulbright scholar. Intrigued by my passion and determination to advance chloroplast genetic engineering, he offered me a long-term position at WSU. From 1984 to 1990, I enjoyed pursuing my dream of chloroplast genetic engineering research and became a permanent U.S. resident ( Figure   1A , B).

Because chloroplasts produce RuBisCO, the most abundant protein on Earth, I imagined that these organelles could serve as bioreactors to make other useful proteins. The concept of chloroplast bioengineering dawned on me with the idea of introducing foreign genes into isolated chloroplasts and reintroduce the modified chloroplasts into protoplasts to regenerate

This article is protected by copyright. All rights reserved "transplastomic" plants. In the mid-1970s, uptake of green chloroplasts by albino protoplasts and regeneration of green or variegated plants had been widely reported (Bonnett 1976; Bonnett & Erickson, 1974) . From then, I invested my creativity and hard work to establish an efficient and prolonged in organello translational system using isolated chloroplasts. At WSU, I used isolated chloroplasts and the cyanobacterium Anacystis nidulans to understand the process of DNA uptake (Daniell et al., 1986b; Daniell & McFadden, 1986; 1988) . It was gratifying to publish the first studies on the uptake and expression of bacterial and cyanobacterial genes by isolated chloroplasts (Daniell & McFadden, 1987) . After reading these publications, Prof. Lawrence Bogorad -then president of the U.S. National Academy of Sciences (NAS) and the American Association for the Advancement of Science (AAAS)-invited me to visit Harvard University on sabbatical leave in 1988. At Cornell University during this period, Prof. John Sanford had designed the Biolistic Particle Delivery System, the so-called gene gun, which we used with my chloroplast vectors to demonstrate the expression of a first foreign gene in chloroplasts (Daniell et al., 1990) . Prof. Bogorad communicated this manuscript to the U.S. Proceedings of the National Academy of Sciences (PNAS) while magnanimously declining authorship and then proudly citing this publication among a dozen key articles in his introduction to the Molecular Biology of Plastids book (Bogorad, 1991) . Our publications in PNAS resulted in the oldest patents on chloroplast genetic engineering (Daniell & McFadden, 1988) and enabled the creation of a first biotech company, Chlorogen inc., based on the use of chloroplast technologies. After three decades of research, vast majority of transplastomic studies use the psbA regulatory sequences that I introduced in our very first chloroplast DNA vector made in 1986 (Daniell et al., 2016a, b) .

Similar to the invention of the gene gun and the creation of chloroplast vectors, an important milestone in the field has been the identification of the aadA gene (Goldschmidt-Clermont, 1991), a selectable marker used for the introduction of more than 300 foreign genes into the chloroplast genome. In contrast to the approach of Prof. Pal Maliga at Rutgers University of inserting foreign genes into transcriptionally silent spacer regions of the chloroplast genome large single-copy (LSC) region (Svab and Maliga, 1993) , I hypothesized that insertion in the transcriptionally active spacer

This article is protected by copyright. All rights reserved regions would utilize the transcription machinery of native chloroplast operons and enhance both the transcription and translation of foreign genes. I could demonstrate the advantages of inserting foreign genes into the inverted repeat region to double the transgene copy number and take advantage of the copy correction mechanism to accelerate homoplasmy (Daniell et al., 1998) . The presence of the chloroplast origin of replication within the flanking sequence provided more copies for integration. These and other concepts developed in my research program, notably related to codon optimization, polycistrons and operons (Kwon et al., 2016) , are now used in several laboratories involved in transplastomic research around the globe (Jin & Daniell, 2015; Daniell et al., 2016 a,b) .

Although the tobacco chloroplast genome was sequenced in 1986, for two more decades only five other crop chloroplast genomes were sequenced. Chloroplast regulatory sequences are species specific, and their sequences or binding proteins are not well conserved. Likewise, not even a single intergenic spacer region is conserved among chloroplast genomes in the grass family, and 100% identical spacer regions are required for transgene integration via homologous recombination (Ruhlman et al., 2010) . In practice, using heterologous regulatory elements for transgene expression is usually not working, as illustrated for instance by the lettuce psbA sequence reducing transgene expression by 80-97% in tobacco, and vice versa for the tobacco sequence in lettuce, when compared to endogenous regulatory elements (Ruhlman et al., 2010) . This prompted us to sequence chloroplast genomes from different crops used in everyday life, including soybean, cotton, potato, tomato, grape, citrus, coffee, carrot, cassava, chickpea, cocoa, peach, chestnut, barley, sorghum, turfgrass (Daniell et al., 2016b) . This field is still lagging behind because <70 genera have sequenced chloroplast genomes among >3,000 cultivated crops.

Conceptual advancement in chloroplast vector design has facilitated the expression of foreign genes in chloroplasts to confer valuable agronomic traits. Stable expression of the Petunia EPSPS gene (provided by Monsanto) via the chloroplast genome conferred high level tolerance to glyphosate when tobacco was sprayed with the commercial herbicide Round-Up. Transgene escape

This article is protected by copyright. All rights reserved via pollen and the possibility of weedy relatives capturing this valuable trait were major concerns at this time, and therefore the integration of a herbicide resistance gene in the chloroplast genome and the demonstration of transgene maternal inheritance was considered then a major accomplishment, an invention that was featured on the cover page of Nature Biotechnology (Daniell et al., 1998) and widely debated in several journals and the public press. Likewise, the development of insect resistance to biopesticides was considered a potential problem due to the low-level expression of truncated Bacillus thuringiensis (Bt) endotoxin proteins in GM crops. By comparison, when a full length B. thuringiensis Cry2Aa2 protoxin gene was expressed in chloroplasts and fed to tobacco budworm, cotton bollworm or beet armyworm, insects with 20,000-40,000-fold resistance to Cry1A or Cry2Aa2 were killed with 100% mortality (Kota et al., 1999) . We also introduced in the chloroplasts the Cry2Aa2 operon, which allowed Bt toxin expression up to 46% of total leaf protein, the highest level of insecticidal protein reported to date in the published literature (DeCosa et al., 2001) . Electron micrographs in this publication showed Bt crystals in chloroplasts similar to those naturally observed during sporulation in B. thuringiensis.

Insects that are difficult to control, including the cotton bollworm and the beet armyworm, were killed after only a single bite of transplastomic leaf tissue expressing the Bt crystals. This paper was featured on the cover of Nature Biotechnology, with the caption "Clear as a crystal." Few years later, we engineered several operons towards remodeling isoprenoid pathways, terpene and artemisinin biosynthesis. Very high levels of the PHBA polymer, reaching up to 26.5% of leaf dry weight, were obtained through this chloroplast metabolic engineering strategy (Vitanen et al., 2004) .

Although these were exciting developments, there was great need to demonstrate these concepts in crop plants, which was not easy however because the regeneration of cereals or legumes following transformation required somatic embryogenesis. We demonstrated the concept of generating transplastomic lines via embryogenesis using non-green carrot cells with the betaine aldehyde dehydrogenase gene inserted into the chloroplast genome. Transplastomic carrot plants grew well in the presence of NaCl concentrations reaching 400 mM, the highest level of salt tolerance reported to date in the published literature (Kumar et al., 2004) . Using similar approaches,

This article is protected by copyright. All rights reserved the Bayer Crop Science group transformed the soybean chloroplast genome to introduce herbicide and insect resistance genes, created marker free transplastomic lines (Dufourmantel et al 2005 (Dufourmantel et al , 2007 and advanced them to field trials. These products were not commercialized because GM soybean using nuclear genetic engineering had already captured the marketplace. Therefore, we focused our efforts on traits that required high levels of expression, unattainable via nuclear genetic engineering. 

Learning valuable lessons from the failure of Chlorogen and unwilling to give up, I persuaded investors of the biotechnology startup PhylloZyme-who also have ownership in the textile and microbial enzyme business-to develop products that required minimal regulatory approval. Most current genetically modified plant commercial products on the market are derived from seeds, and therefore it was quite thrilling to launch a first leaf-made protein product for commercial use Accepted Article developed by Phyllozyme in the summer of 2018 (Daniell et al., 2019c; Kumari et al., 2019) . Dry leafpectinases were validated with eight liquid commercial microbial-enzyme products for textile or juice industry applications. In contrast to commercial liquid enzymes requiring cold-storage and transportation, leaf-pectinase powder could be stored up to 16 months without any protective formulation at ambient temperature without loss of enzyme activity. Natural cotton fiber does not absorb water due to the hydrophobic nature of waxes and pectins. After bioscouring with pectinase, they readily absorbed water in a few milli-seconds, meeting industry requirements.

Similarly, four newly launched leaf-enzyme products (endoglucanase, exoglucanase, lipase, mannanase) were also compared and validated with 15 commercial microbial-enzyme products for the detergent and textile industries (Kumari et al., 2019) . Leaf-lipase/mannanase crude-extracts removed chocolate or mustard oil stains effectively at both low and high temperatures.

Endoglucanase and exoglucanase in crude leaf-extracts removed dye efficiently from denim surfaces and depilled knitted fabric by removal of horizontal fiber strands. Thus, the Phyllozyme leafproduction platform offers a novel low-cost approach by the elimination of fermentation, purification, concentration, formulation, and cold-chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.

While non-therapeutic proteins/enzymes are advanced quickly to the market, the timeline for advancing therapeutic products are much longer. The first vaccine antigen (CTB) was expressed in chloroplasts two decades ago , followed by several other vaccine antigens to treat malaria, tuberculosis, HIV, dengue, anthrax plague etc. and their efficacy have been evaluated using pathogen or toxin challenges (Chan & Daniell, 2015) . More recently, vaccine antigens against infectious diseases have been evaluated by the FDA and CDC, supported by the Gates Foundation.

Oral delivery of chloroplast-made polio viral protein 1 (or VP1) antigen common to all polio serotypes conferred both mucosal and systemic immunity, generating both IgA and IgG1 antibodies specific to VP1. Polio virus neutralization studies performed at the CDC, using hundreds of sera

This article is protected by copyright. All rights reserved transmitted. There are >50 vaccine clinical trials in progress that will primarily produce systemic but not mucosal surface immunity that is required to protect at the surface of viral entry. In addition, waning of antibody response (as seen already in repeat Covid-19 infection) or poor immune response in elderly patients would require multiple oral boosters and scale up capacity to vaccinate the global population. Therefore, my lab is now developing booster vaccine to assure long term mucosal immunity in elderly patients and provide affordable protection against reinfection for mass affordable vaccination to deal with Covid-19 pandemic that has devastated global health and economy.

Over the last fifty years, injections of recombinant human insulin made in yeast or bacteria save millions of lives, but these products are not affordable for more than 90% of the global diabetic population. Insulin pumps cost $6,000-12,000 while one-third of the global population earn less than $2 a day. Therefore, several human therapeutic proteins have been expressed in chloroplasts to develop affordable protein drugs to treat diabetes, hypertension, heart failure, retinopathy, hemophilia or Alzheimer's disease (Daniell et al 2016a; 2019a) . Protein drugs can be produced in lettuce chloroplasts in cGMP facility and they are stable in lyophilized plant cells for several years when stored at ambient temperature, eliminating the cold chain for storage and transportation (Su et al 2025). The plant cell wall efficiently protects vaccine antigens from digestive enzymes and acidic pH, and gut microbes digest plant cell walls to release antigens in the gut lumen. Recent advances include expression of human blood proteins after codon optimization (Kwon et al 2016 (Kwon et al , 2018 in marker free lettuce plants, stability of expression in the absence of antibiotic resistance genes and functional efficacy in animal disease models Daniell et al 2020) . In Accepted Article addition to raising the profile of plant biology in the medical field by featuring plant research on covers of medical journals or on featured editorials, these projects received funding from Bayer, Novo Nordisk, Shire, Takeda and NIH SMARTT program to produce clinical grade materials, conduct third party toxicology/ pharmacokinetic/regulatory studies at Stanford Research Institute and results of these studies have been published recently (Herzog et al 2017; Daniell et al 2020) .

The SARS-CoV-2 spike protein binds to ACE2 with high affinity, infects alveolar cells, results in lung injury, and dramatically lowers ACE2 levels. In healthy human lungs, ACE2 produces surfactants to protect alveoli from collapsing and cytoprotective anti-inflammatory Ang (1-7) peptide. Thus, ACE2 supplementation is beneficial by serving as a decoy to reduce SARS-CoV-2 entry into human cells and by protecting against lung injury via the anti-inflammatory actions of Ang (1-7).

My lab has produced clinical-grade oral ACE2. Oral delivery results in 10-fold higher concentration in lungs and prevents and treats pulmonary hypertension at doses with no evidence of toxicity . Therapeutic efficacy and safety of supplementing ACE2 and Ang (1-7) with this existing product in non-critically ill COVID-19 patients in the hospital and at home is in progress. By employing an integrated Phase 1 and 2 clinical trial design, this clinical trial addresses an urgent unmet medical need to treat the growing population of COVID-19 patients and protect them from lung and extra-pulmonary tissue injury.

As a plant biologist, I consider myself extremely fortunate to be teaching at an Ivy League University, featured among the most eminent scholars of this institution by President Amy Gutmann at the Power of Penn, London (Figure 1C) 

This article is protected by copyright. All rights reserved I am grateful to Prof. Laurence Bogorad for inviting me on sabbatical, paying for the Harvard Radcliff guest house, communicating my manuscript to PNAS and declining authorship in order to promote a foreign young scientist to lead this field. Dr. Andrew Marshall, Editor-in-Chief of Nature Biotechnology, believed in this concept when it was shunned by many in this field, featuring chloroplasts several times on the journal's cover and ranking my research among the top ten inventions of the decade in 2007. Likewise, I am grateful to biomedical journals such as Blood, Hypertension and Molecular Therapy for not only publishing our plant biology articles, but also writing featured editorials, highlighting our work on the cover or granting us 'best paper' awards. Jihong-Liu Clarke, I also had the opportunity of organizing the first conference on chloroplast biotechnology in 2006, that was attended by more than 300 participants from 20 countries worldwide. It was amazing to see this new field of research pioneered in my lab pursued by so many Accepted Article investigators worldwide and gratifying then to see the output of this first conference giving rise to a new, and still continuing, Gordon Research Conferences series on chloroplast biotechnology. I was the recipient of the highly coveted campus-wide Sigma XI Award (Auburn University) and Pegasus Professorship, Board of Trustees Chair for best teaching, research and service ( Figure   1D ) and addressing the commencement ceremony (Figure 2A) 

plant biology, and for this reason I am raising awareness of racial inequalities among foreign born plant scientists in the United States. Finally, scientific journals are acknowledging racial discrimination rampant for decades (Cell Editorial, 2020) .

After many years of experience in science, my greatest satisfaction is to see many of my mentees lead multi-disciplinary research programs in medical schools, industry, academia or federal agencies, including the West Wing of the White House as Presidential Innovation Fellows. I am proud to share photos of my lab colleagues at Auburn, University of Central Florida and Pennsylvania (Figures 3 A-D) , who made significant contributions to advance this field. My prayer for my mentees is not to get discouraged by institutional racial bias or discrimination but to believe in the power of persistence over prejudice. In my life, American institutional bias was compensated by European institutions and noble Americans. Most importantly, passion for one's career and commitment will overcome adversities. My passion for chloroplasts has lasted for four decades, and I can't stop thinking about new opportunities for chloroplast genetic engineering. When I grew up in India, I experienced first-hand the horrors of people dying without access to medicines or vaccines. Therefore, I am deeply committed to advancing my inventions to the clinic and to make affordable protein pharmaceuticals for the global population. I am thankful to my research team members for their hard work, and to my wife Shobana and my sons, Luke and Paul, who made great sacrifices to help me realize this goal.

Introduction

Mechanism of uptake of Vaucheria chloroplasts by carrot protoplasts treated with polyenthylene glycol

Accepted Article This article is protected by copyright. All rights reserved

Transfer of algal chloroplasts into protoplasts of higher plants

Science has a racism problem

Plant-made oral vaccines against human infectious diseases -Are we there yet?

Cold chain and virus free chloroplast made booster vaccine to confer immunity against different polio virus serotypes

Vaccination through chloroplast genetics: affordable protein drugs for the prevention and treatment of inherited or infectious diseases

Containment of herbicide resistance through genetic engineering of the chloroplast genome

An efficient and prolonged in vitro translational system from cucumber etioplasts

Plant cell-made antigens for induction of oral tolerance

Expression and assembly of the native cholera toxin B subunit gene as functional oligomers in transgenic tobacco chloroplasts

Chloroplast genomes: diversity, evolution and applications in genetic engineering

Investigational new drug enabling angiotensin oral-delivery studies to attenuate pulmonary hypertension

Accepted Article This article is protected by copyright. All rights reserved

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts

Genetic enginerring of plant chloroplasts

Cold-chain and virus free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes

Validation of leaf and microbial pectinases: commercial launching of a new platform technology

Transformation of the cyanobacterium Anacystis nidulans 6301 with the Escherichia coli plasmid pBR322

Transient foreign gene expression in chloroplasts of cultured tobacco cells following biolistic delivery ofchloroplast vectors

Hyper-expression of the Bt Cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

Generation and analysis of soybean plastid transformants expressing Bacillus thuringiensis Cry1Ab protoxin

Generation and Characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance. Accepted Article This article is protected by copyright. All rights reserved

Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas

Oral tolerance induction in hemophilia B dogs fed with transplastomic lettuce

The engineered chloroplast genome just got smarter

Ovexpression of the Bacillus thuringiensis Cry2A protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

Plastid expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots and leaves confers enhanced salt tolerance

Validation of leaf enzymes in detergent and textile industries: launching of a new platform technology

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice

Codon optimization to enhance expressions yields insight into chloroplast translation

Oral delivery of novel

I would like to express thanks to several colleagues at Penn and Drs. Marc Cohn (LSU), Dominique Michaud (Laval Univ) for their valuable comments on this manuscript.