A Genetic Engineering Strategy to Eliminate Peanut Allergy Hortense Dodo, Phd, Koffi Konan, Phd, and Olga Viquez, Phd

A Genetic Engineering Strategy to Eliminate Peanut Allergy Hortense Dodo, PhD, Koffi Konan, PhD, and Olga Viquez, PhD

Address approximately 2% of the population is affected [1]. Food Food Biotechnology Laboratory, Department of Food & allergy is defined as an allergy that occurs when the immune Animal Sciences, PO Box 1628, Alabama A&M University, Normal, AL system mistakenly responds defensively to a specific food 35762, USA. E-mail: [email protected] protein that is not harmful to the body. Peanut is the most common cause of severe or fatal Current Allergy and Asthma Reports 2005, 5:67–73 Current Science Inc. ISSN 1529-7322 food-associated anaphylaxis. Approximately 50% of the Copyright © 2005 by Current Science Inc. annual hospital emergency department visits and approximately 63% to 67% of deaths due to anaphylaxis [2] are caused by peanut. Peanut allergy is an IgE-mediated hyper- Peanut allergy is an IgE-mediated hypersensitivity reaction sensitivity reaction [3] that is acute with severe symptoms, with an increasing prevalence worldwide. Despite its life threatening, and outgrown by only few, unlike allergy to seriousness, to date, there is no cure. Genetic engineering egg and cow’s milk [4]. It was estimated that 1.1% of the US strategies can provide a solution. The post-transcriptional population or approximately 4.4 million children and gene silencing (PTGS) model can be used effectively to knock adults suffer from allergy to peanut and tree nuts [5]. Symp- out the production of allergenic proteins in peanut by specific toms associated with peanut allergy vary from swelling of degradation of the endogenous target messenger RNA the lips or tongue, vomiting, difficulty breathing, and loss of (mRNA). Ara h 2, the most potent peanut allergenic protein, consciousness to anaphylactic shock leading to death. was selected as a model to demonstrate the feasibility of this Accidental ingestion of peanuts in unsuspected food is concept. Transgenic peanut plants were produced via increasing because of undeclared peanut and its ubiquitous microprojectile–mediated transformation of peanut embryos use as an ingredient in processed foods. The exposure to using a plasmid construct, which contains a fragment of the exotic foods that contain peanut is also a source of accidents. coding region of Ara h 2 linked to an enhanced CaMV 35S Several research teams have proposed a variety of constitutive promoter. Molecular analyses, including approaches to reduce or eliminate peanut allergy, including polymerase chain reaction and Southern blots, confirmed the thermal processing, immunotherapy, and anti-IgE and DNA presence of the stable integration of the Ara h 2 transgene vaccines [6–11]. However, to date, there is still no cure. Epi- into the peanut genome. Northern hybridization showed the nephrine and antihistamines are only administered to expression of the Ara h 2 transgene in all vegetative tissues of reverse the symptoms. Currently, the most effective manage- the mature transgenic peanut plants, indicating the stable ment option available to the susceptible population is to expression of the truncated Ara h 2 transgene throughout avoid peanuts and peanut-containing products. However, as the development of the plants. It is, therefore, reasonable to mentioned, avoiding peanut has proven difficult, particu- expect that the truncated Ara h 2 transgene transcripts will larly owing to undeclared peanut contents. be synthesized in the seeds and will trigger the specific degra- However, peanut is a highly nutritious, popular, and dation of endogenous Ara h 2 mRNA. The next step will be inexpensive food worldwide. It is the number 1 snack nut to grow the transgenic peanut plants to full maturity for seed consumed in the United States and accounts for two thirds production and to determine the level of allergen Ara h 2. of the total snack-nut market. Peanut contributes more than 4 billion dollars annually to the US economy. It is a legume rich in proteins, monounsaturated and polyunsaturated Introduction fatty acids, carbohydrates, and fibers. It is also a good source Food allergy is a rapidly increasing phenomenon in indus- of vitamins E, B1, B2, B6, folic acid, thiamin, niacin, and trial countries. It has been reported that every 10 years there minerals, such as copper, manganese, phosphorous, iron, is as much as a 25% increase in food allergy in children as magnesium, calcium, selenium and zinc. Peanut has been well as in adult populations [1]. Food allergy is the leading linked to many health benefits. For example, it has the cause of anaphylaxis treated in US hospital emergency potential to lower blood cholesterol and reduce the risk for departments. It is estimated that in the United States, food heart diseases because of its content of the heart healthy allergies affect 6% to 8% of children and 1% of the adult mono-unsaturated fat and resveratrol. Peanut is also a good population. In France, 5%, and in the United Kingdom, source of folic acid, which helps prevent neural tube defects, 68 Food Allergy

Table 1. Comparative characteristics of cDNA and genomic clones of peanut allergens Ara h 1, Ara h 2, and Ara h 3 Homology: Number of Coding region Introns, genomic vs amino acid Allergen cDNA, bp genomic clones, bp number/size, bp Promoter, bp cDNAs, % residues in protein Ara h 1* 1878§ 1878 Intron 1/71 1926 100§ 626* 1842¶ 1878 Intron 2/249 1926 98¶ 626§ 1878 Intron 3/74 1926 614¶ Ara h 2† 333** 621 No introns 109 98** 207† 516†† 621 No introns 109 97†† 111** 507‡‡ 621 No introns 109 95‡‡ 172†† 621 No introns 109 169‡‡ ARA h 3‡ 1521§§ 1614 Intron 1/339 660 96§§ 538‡ 1590¶¶ 1614 Intron 2/87 660 95¶¶ 507§§ 1614 Intron 3/168 660 530¶¶

*,†,‡—Genomic clones GenBank Accession #AF432231, #AY0072, and #AF5108. §,¶—Ara h 1 cDNAs GenBank Accession #L34402 and #L38853. **,††,‡‡—Ara h 2 cDNAs GenBank Accession #L77197, #AY158467, and #AY117434. §§,¶¶—Ara h 3 cDNAs GenBank Accession #AF093541 and #AF086821. bp—base pairs; cDNA—copy DNA. and phytosterols, which may offer protection from colon, The first generation of agricultural products resulting prostate, and breast cancer [12]. from GM focused largely on input agronomic traits, such as disease-resistant Bacillus thuringiensis (Bt) corn, soybean, and cotton, and herbicide-resistant crops. The next genera- Genetic Modification: A Promising Strategy to tion will focus more on value-added output traits, such as Alleviate Peanut Allergy nutritional quality as seen in the pro-vitamin A–enriched What is genetic modification? golden rice, and the hypoallergenic peanut, discussed later. Plant genetic engineering is a relatively recent technology with enormous benefit for the field of agriculture. It offers Peanut allergens are seed storage proteins an efficient and cost-effective means to produce a wide Peanut contains 25% to 35% proteins that are seed storage array of novel, value-added plant and food products in an proteins [12]. Peanut proteins are composed of the two environmentally friendly manner. It is becoming increas- major globulin families of storage proteins known as ingly important worldwide because it provides significant arachin (legumin) and conarachin (vicilin) [13]. They improvements in the quantity, quality, and acceptability of accumulate in the developing seeds and are hydrolyzed the world's food supply and may be the best source for during germination as a source of amino acid and nitrogen food security in developing countries. for the growing seed [14]. Genetic engineering is also referred to as genetic modifi- A protein profile of peanut extract shows several protein cation (GM), molecular biology, or recombinant DNA tech- bands, out of which 6 to 19 react with human IgE and nology. A genetically modified organism (GMO) is defined induce an immunogenic response in susceptible people as an organism whose genetic make-up has been modified [15,16]. Peanut allergens constitute approximately 5% of by scientists to achieve a specific, targeted goal. Genetic engi- the total protein of a cell. They are heat stable and resistant neering tools are enabling scientists to pursue exciting novel to digestion [17]. strategies for plant improvement, most of which would be Six peanut allergens have been identified, and three of impossible using traditional plant-breeding methodologies. them have been extensively characterized at the protein, Genetic modification is an improvement from conven- copy DNA (cDNA), and genomic DNA levels. They are Ara tional plant breeding because it increases the precision of h 1 (vicilin), Ara h 2 (conglutin), and Ara h 3 (glycinin). gene transfer (only selected genes with the desirable traits The first two are generally defined as major allergens are transferred to the recipient plant), and the time to because they are recognized by the serum IgE of more than obtain the desirable plant is reduced. Conventional plant 90% of a group of susceptible individuals [18]. breeding is less controlled, more random, and results in Ara h 1 was the first major peanut allergenic protein to the transfer of several unknown and unwanted genes. With be isolated [19]. It is a glycoprotein of 63.5 kDa, with an this new technology, genes from different species and king- isoelectric point of 4.55 and two isoforms identified as doms can be interchanged from one organism to another, clone P17 (1949 base pairs [bp]) and clone 41B (2032 bp), a particularity that makes the technology controversial. with 96% similarity at the cDNA level [20] (Table 1). The A Genetic Engineering Strategy to Eliminate Peanut Allergy • Dodo et al. 69

Figure 1. Schematic representation describing the flow of genetic information. A, The Central Dogma of Molecular Biology: a gene is transcribed into a messenger RNA, which is translated into a protein. B, Schematic representation highlighting key steps in silencing peanut-allergen genes based on the PTGS model using peanut allergen Ara h 1 as an example. dsRNA—double-stranded RNA; PTGS—post-transcriptional gene silencing; RISC—RNA-interfering silencing complex; RNase—ribonuclease.

genomic clone of peanut allergen Ara h 1 was isolated in 72% homology with glycinins (legumins) from soybean our laboratory [21]. It is a full-length gene with an open and pea. The genomic Ara h 3 clone was also isolated in our reading frame (ORF) of 2275 bp and 3 introns of 71,249, laboratory. It is a 3.5 kb clone with 3 introns and a promoter and 74 bp. Over 2000 bp of the promoter have been of 660 bp [27]. Ara h 3 was shown to possess trypsin inhibi- sequenced and contain several features found in strong tory activity and to share high nucleotide sequence homol- plant gene promoters. ogy with trypsin inhibitors [28]. Two isoforms have been Ara h 2 is the smallest but the most potent among the identified for Ara h 3 (Table 1). three common allergens [22]. It is a 17.5-kDa glycoprotein Kleber-Janke et al. [16] described three additional aller- with an isoelectric point of 5.2 [23]. The protein is less gens, Ara h 5 (profilin) and Ara h 6 and Ara h 7 (both con- abundant in the seed than Ara h 1 and perhaps less essential glutin-homologue proteins). These allergens are defined as for normal seed morphology and function. Ara h 2 is also minor allergens because they are recognized by serum IgE reported to function as a trypsin inhibitor [24]. A partial of less than 50% of peanut-susceptible individuals [16]. cDNA sequence for Ara h 2 of 460 bp was published [18]. The genomic clone that encodes the messenger RNA ([mRNA] cDNA) was isolated in our laboratory [25••]. The Post-transcriptional Gene Silencing to full-length genomic clone has an ORF of 621 nucleotides, a Produce a Hypoallergenic and/or deduced amino acid content of 207 residues, and a putative Allergen-free Peanut signal peptide of 21 amino acid residues. Comparison of the Central dogma of molecular biology genomic and cDNA sequences of Ara h 2 reveals the absence The flow of genetic information dictates that “DNA is tran- of an intron and the presence of at least three isoforms of scribed into a[n] RNA that is translated into a protein” (Fig. the same gene family in the peanut genome (Table 1). 1). Peanut allergens are produced using this concept. For Ara h 3 encodes a 60 kDa preproglobulin (preprolegu- example, in the case of peanut allergen Ara h 1, the genomic min; arachin polypeptide). It is considered a minor allergen clone of 2275 bp is transcribed into the Ara h 1 mRNA relative to Ara h 1 and Ara h 2 because it is recognized by less (1878 bp), which is translated into the Ara h 1 allergenic than 50% of serum IgE of peanut-allergic individuals [26]. protein of 626 residues (Fig. 1A). The coding region of the Ara h 3 cDNA clone is 1521 nucle- When peanut is eaten, allergenic proteins are recognized otides long, coding for 507 amino acids that share 62% to as antigens in peanut-sensitive individuals. For example, the 70 Food Allergy

Ara h 1 antigen binds to the antigen-binding sites of an IgE endogenous peanut DNA for expression. Transcripts from antibody, which triggers the mast cells to which it is attached the transgene initiate long dsRNA molecules, which would to degranulate and release a variety of active agents, includ- be processed into small 21 to 25 nucleotides (siRNAs) by an ing histamine and leukotrienes. It is the release of these sub- endogenous RNAIII enzyme called “Dicer” [40]. These stances into the surrounding tissue that causes local or siRNAs would guide the RNA-interfering silencing complex systemic allergic reactions. (RISC), which contains the proteins necessary for unwinding To bring a solution to peanut allergy, one can consider the double-stranded siRNAs, and cleave the endogeneous either 1) altering the immune response of the allergic indi- Ara h mRNAs at the site where the antisense RNAs are bound viduals or 2) a less invasive approach—that is, to knock [41]. Sources of dsRNA formation from Ara h transgene tran- out the production of the allergenic proteins. scripts include: 1) pairing of transcripts transcribed from an Blocking the production of allergens can be done by inter- inverted repeat (IR) transgene; 2) pairing of the normal sense fering with the flow of the genetic information (Fig. 1B). For RNAs and antisense RNAs (asRNA) arising from aberrant example, to eliminate the production of the Ara h 1 protein, transcription of the transgene; 3) pairing of complementary we could interfere either at the mRNA transcription level regions of RNA degradation products; and 4) pairing of tran- (transcriptional gene silencing [TGS]) or at the post-transcrip- scripts with antisense RNA (asRNA) produced by RNA- tional level (PTGS). The PTGS was adapted for silencing pea- directed RNA polymerase (RdRP) [42,43]. nut allergens because of its specificity for mRNA degradation. Steps involved in silencing peanut-allergen genes Principle of post-transcriptional gene silencing Engineering a hypoallergenic or allergen-free peanut requires The advancements made in genetic engineering have led to the following steps: 1) isolation of the endogenous allergen the possibility of knocking out the production of specific genes; 2) construction of the transformation cassettes using proteins in organisms by downregulating and/or silencing allergen-gene fragments as transgenes; 3) transformation of the genes encoding these proteins. Strategies developed to transgenic peanut via biolistic or Agrobacterium tumefaciens downregulate genes in plants include mutation-based reverse and regeneration; 4) molecular analyses to identify trans- genetics [29], gene targeting [30], antisense RNA [31], co- genic peanut lines; 5) immunologic analyses of transgenic suppression [32,33], and the newly uncovered RNA inter- seeds for allergen reduction; and 6) nutritional analysis for ference (RNAi) [34]. Genetic and biochemical evidences sug- quality determination. gest that antisense-mediated gene silencing, co-suppression, and RNAi are all inputs into a common RNA silencing pathway triggered by the formation of a double-stranded RNA Isolation and Cloning of Ara h Genes (dsRNA). This pathway, called PTGS, is characterized by A prerequisite to silencing peanut-allergen genes is the acqui- accumulation of 21 to 25 nucleotides, small-interfering RNAs sition of DNA fragments encoding the target proteins and (siRNAs), sequence-specific degradation of target mRNA, their use as transgenes in the construction of transformation and methylation of target gene sequences [35]. cassettes. Although cDNA clones of peanut allergen genes Most allergen proteins in plants are present as isoforms were published [18,19,26], we decided to isolate and encoded by genes that are members of multigene families sequence the genomic clones of target allergens for several [36,37]. In peanut, as shown in Table 1, several isoforms reasons: 1) to better understand the structure and regulation have been identified for Ara h 1, Ara h 2, and Ara h 3 of the genes; 2) to uncover information not available at the [21,25••,38,39]. To reduce or to eliminate the production of cDNA level, such as presence of intron and promoter region; these allergens, it will be more efficient to target several 3) to identify and isolate intron DNA fragments that can be genes at once. PTGS technologies, such as the use of anti- used in the construction of transformation cassettes. sense or RNAi-based constructs that selectively inhibit gene expression by targeting sites on mRNA transcribed from the Construction of transformation vectors selected genes, will provide effective approaches to eliminate Peanut allergen transgenes allergens from peanut. High-sequence homologies exist between peanut-allergen Post-transcriptional gene silencing is initiated by dsRNA isoforms. Homologous regions will be PCR-amplified and molecules that mediate the degradation of homologous tran- used as transgenes to silence all peanut-allergen isoforms. scripts. Figure 1B gives a schematic representation of the PCR products can be mounted as sense, antisense, or IR molecular process that would be involved in the degradation transgenes. Because of the dominant silencing properties of of mRNA transcripts from peanut allergen (Ara h) genes, IR transgenes, they have been more exploited for research based on the PTGS model. A fragment of the Ara h gene is purposes and practical applications. However, although amplified by polymerase chain reaction (PCR) and cloned phenotype suppression by IR, co-suppression, or antisense into a transformation vector. This vector is a DNA vehicle, RNAs can persist in vegetatively growing plants, and in some which transfers the Ara h gene fragment (called transgene) cases, be meiotically transmitted to progeny, silenced genes into peanut cells following biolistic or Agrobacterium- can return to an expressed state, indicating that the stable mediated transformation. The transgene integrates into the changes are potentially reversible [44]. For crop plant A Genetic Engineering Strategy to Eliminate Peanut Allergy • Dodo et al. 71 improvement, Stoutjesdijk et al. [45••] reported stable phe- Silencing Peanut Allergen Ara h 2 notype suppression induced by hairpin RNA (hpRNA) Construction of transformation cassette and transgene constructs. In an hpRNA transgene construct, the transformation of peanut embryogenic tissues target gene is cloned as an inverted repeat spaced with an Ara h 2 is the most offending allergenic protein in peanut unrelated sequence. When an intron is used as the spacer [52]. Therefore, elimination of this allergen would be a criti- (intron hairpin RNA construct [ihpRNA]), the efficiency of cal step in the process of engineering a hypoallergenic and/ silencing becomes very high in terms of both the proportion or allergen-free peanut variety. The elimination of Ara h 2 of independent transgenic plants showing silencing (almost alone will significantly reduce the allergenicity of peanut, 100%), and the degree of silencing [46•]. This suggests that and demonstrate proof of the concept of silencing peanut ihpRNA constructs made of peanut-allergen transgenes are allergen genes. the most appropriate for the development of peanuts with A 430 bp fragment was PCR-amplified from the coding reduced allergenicity. Intron regions identified in peanut region of Ara h 2 genomic DNA and used to construct a genomic clones can be used in the construction of the trans- transformation vector. The truncated Ara h 2 transgene was formation cassette. flanked by an enhanced version of the constitutive CaMV 35S promoter, and by the nopaline synthase terminator Promoter (NOS term) of A. tumefaciens, and cloned into the pUC18 Promoters are DNA regions located just upstream of the vector. The resulting pDK2 plasmid was co-bombarded gene sequence. Promoters contain the binding sites for with plasmid pCB13, a selection plasmid harboring the RNA polymerase and are, therefore, crucial to the expres- hygromycin-resistant gene to transform peanut embryo- sion of the genes. They also contain several regulatory ele- genic tissues. Five independent bombardment experiments ments that act as binding sites for distinct proteins, known were performed using embryogenic callus initiated from as transcription factors. The previous binding of these tran- the Georgia green peanut variety [53••]. scription factors allows the RNA polymerase to be loaded onto the promoter so it can begin the process of transcrip- Regeneration of transgenic peanut plants containing tion. Because peanut allergens are seed proteins, a pre- the Ara h 2 transgene and molecular analyses requisite for the preparation of transformation vector is the Two to 3 months after co-bombardment of pDK2 and the use of a legume seed–specific promoter to eliminate aller- selection plasmid pCB13, 48 independent hygromycin- gens during seed development. Alternatively, a strong con- resistant callus lines were regenerated. PCR analyses were stitutive promoter, such as the CaMV 35S, can be efficiently performed to identify cell lines containing the CaMV 35S used to achieve the same goal. promoter, a DNA fragment not naturally present in the peanut genome. Approximately 75% (36/48) callus lines Modes of transformation showed the 0.6 kb band, corresponding to the size of the Protocols for biolistic and Agrobacterium-mediated transfor- CaMV 35S promoter, and transgenic plants were regener- mation have been developed for peanut. The biolistic- ated from these positive callus lines within 9 to 13 months mediated transformation [47] is currently one of the most after bombardment [53••]. widely used because it has the advantage to overcome the The stable integration of the Ara h 2 transgene into the genotype barrier, and to make it possible to transform differ- peanut genome was confirmed by Southern hybridization. ent peanut genotypes. Mature peanut embryonic axes are The target sequence was the CaMV 35S promoter. DNA was removed from seeds and cultured on Murashige and Skoog extracted from both transformed and non-transformed cal- (MS) medium [48] containing 3 mg/L picloram to initiate lus lines, and digested with HindIII/BglII to release the 0.6 somatic embryos. Embryogenic tissues are subcultured for kb promoter fragment. The hybridization probe was a PCR tissue increase, and to produce homogenous tissues before product of the CaMV 35S promoter. The hybridization transformation. A plasmid containing a selection marker bands show the stable integration of the transgene in the gene, such as the hygromycin gene, is co-bombarded with peanut genome [53••]. the plasmid containing the gene of interest for the selection A Northern hybridization experiment was performed of transgenic plants. This protocol takes at least 10 months on total RNA extracted from callus, roots, petioles, and to produce a transgenic plant. leaves of transgenic and non-transgenic plants. The hybrid- Alternatively, several research groups have reported A. ization probe was the 32P-labeled, 430-bp, Ara h 2 trans- tumefaciens-mediated transformation protocols to produce gene fragment. Hybridization bands were detected in fertile transgenic peanut plants within 5 to 7 months transgenic callus lines, but no band was observed in the [49,50]. However, genotype and/or tissue specificity of control non-transformed callus and callus transformed Agrobacterium strains is a limitation to the success of trans- with the selection plasmid pCB13 only. In addition, tran- formation [51]. Our preliminary experiments using Agro- script accumulation from the Ara h2 transgene was also bacterium strain EHA105 to transform hypocotyls of two observed in the leaves and the roots of transgenic peanut peanut varieties, Georgia green and Valencia (New Mex- plants [53••]. In a comparative hybridization experiment, ico), have shown good reproducibility. the same probe was used to detect endogenous Ara h 2 72 Food Allergy

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