Thus, in one embodiment a sub-unit of the assembly unit performs oligonucleotide synthesis, for example with an oligonucleotide synthesizer. A robotic arm can be included to perform transfer of the reaction container e. In one emobodiment the system is provided with already synthesized oligonucleotides and assembles the oligonucleotides into one or more DNA molecules. In each case the sub-units are prepared with the necessary reagents, chemical, and biological building blocks to perform the function in an automated manner. Thus, the assembly unit can begin to perform one or all of the functions immediately upon being provided with the biological sequence information from the receiving unit.
The assembly unit can thus in an automated fashion direct the synthesis of the desired biological entity. All of the units in the system, including the assembly unit, can be prepared with necessary software and pre- charged with necessary reagents and chemicals in the required vessels so that the biological sequence information can be received by the receiving unit and the assembly unit can instantly begin synthesizing the desired biological entity. Thus, the assembly unit can be prepared for assembly or synthesis of the biological entity prior to receiving the biological sequence information.
The systems and methods can also assemble DNA molecules from oligonucleotides. Thus, the nucleic acid molecule or protein or peptide can be assembled through a series of reactions. The oligonucleotides can also be assembled into double-stranded nucleic acid molecules. Persons of ordinary skill are also aware of methods of assembling peptides into polypeptides and proteins, which can also be applied in the invention. The vessels can be containers made of glass or another suitable material and can be pre-filled by the user prior to the receiving unit receiving the biological sequence information from the transmitting unit.
Thus, the assembly unit can be set up and waiting to receive the biological sequence information from the transmitting unit. In some embodiments the functions of the assembly unit or sub-sets thereof will be managed and orchestrated by appropriate software that will direct the combination of materials from the various vessels.
Each sub-unit of the assembly unit can operate from its own software upon receipt of the required information or the assembly unit can have one program that directs the various sub-units that may comprise the assembly unit. In different protocols extracts of rabbit reticulocytes, wheat germ, E. Human in vitro translation systems can be useful when the biological entity is a vaccine for use against human pathogens. The extracts can be prepared and contain the essential components of cellular translational machinery such as, for example, ribosomes, tRNAs, aminoacyl-tR A synthetases, initiation, elongation, and termination factors, energy sources such as ATPs, necessary co-factors and other proteins.
In vitro transcription and translation reactions can be performed at great speed and at the microliter or nanoliter level. A variety of commercially available kits can also be used for convenient setup and have been used with success but the essential components can also be assembled generically.
Whether kits or generic materials are used, in some embodiments a cell-free solution containing essential components of the cellular translational machinery is utilized. Some systems utilize proprietary accessory proteins, ATP, and an energy regenerating system to sustain the synthesis of target proteins from DNA templates.
The extracts can be prepared and pre-charged into a vessel of the assembly unit and be available to perform translation of synthesized nucleic acid when needed. The assembly unit can maintain these extracts at required temperatures to extend their useful life. Upon transcription of RNA a portion of the reaction can be transferred into the vessel for performing translation, or the transcription and translation reactions can be performed in a single vessel.
In some embodiments the biological entity requires protein modifications, such as glycosylated residues, a refolding step, or enzymatic modifications that must occur prior to the biological entity having activity or full activity. Reagents and programming protocols can be included within the assembly unit for this purpose as well. The systems of the invention can also include sub-units, reaction areas, or reaction zones for the purification of biological products. The system has an assembly unit for assembling the biological entity according to the provided biological sequence information.
The assembly unit contains or is connected to vessels containing biological building block molecules or building block polymers and has components for transporting reagents within the system and for executing steps in an automated method of the invention for synthesizing the functional biological entity. These systems can also have any of the components or features described herein. The biological entity can be any as described herein e. The system can have a receiving unit for receiving a signal or data having the biological sequence information. The biological sequence information can be provided by manually keying in the sequence via an electronic interface, or by transmitting the sequence to a receiving unit from a transmitting unit, as described herein.
In some embodiments the biological entity is a nucleic acid or a DNA molecule. When the biological entity is a DNA molecule it can be a functional DNA molecule, meaning that it can be transcribed into an RNA that can be translated into a functional protein or peptide, or is directly useful as a DNA molecule e.
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A functional RNA molecule can be translated into a functional protein or peptide. A functional protein or peptide is one that has a clinical use in the treatment of a disease or disorder, or is directly useful in some biological context e. In various embodiments the functional protein or peptide can be the domain of a protein, a binding subunit, an enzyme or enzyme subunit that has enzymatic activity, a protein or peptide that has antigenic activity that is useful in the generation of a vaccine, a viral protein or a subunit thereof, a viral coat protein e.
The clinical use can thus be the generation of an antigenic response or the binding of a protein or peptide to a specific binding molecule or receptor but in one embodiment the antigenic response is one that furthers the treatment of a disease or disorder e. The functional protein or peptide can also provide a desirable property such as, for example, a desirable taste, texture, scent, or another property. In one embodiment a functional protein or peptide has a function other than the generation of an immune or antigenic response. Oligonucleotides of up to about bp can be assembled but greater accuracy can often be achieved by synthesizing smaller oligonucleotides.
In various embodiments the biological entity is a single-stranded or double-stranded DNA molecule of greater than base pairs in length, or greater than bp, or greater than bp, or greater than bp, or greater than bp, or greater than 1 kb, or greater than 2. Using additional techniques the person of ordinary skill with resort to the present disclosure will realize that DNA sequences synthesized as oligonucleotides can also be joined to assemble a DNA molecule using the method described herein of greater than 1 kb, or greater than 2 kb, or greater than 3 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb.
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In other embodiments the system and method of the invention can be used to assemble a DNA molecule of greater than kb, or greater than kb, or less than kb, or greater than kb, or less than kb, or greater than kb, or greater than kb or greater than 1 mega-base or less than 1 mega-base. In some embodiments the biological sequence information is the sequence or order of nucleotides, amino acids, or other building block that comprises the primary structure of the nucleic acid or peptide or protein that is the biological entity.
In different embodiments this information may be provided to the receiving unit in a binary form or in another encoded form. Thus, in different embodiments the biological entity is a DNA molecule or an RNA molecule and the biological product can be any biological product made therefrom such as, for example, a viral genome or a portion thereof, a viral particle or viral coat or a portion of either, a bacteriophage or portion thereof, an antigenic portion of a viral particle or viral coat, a bacterial genome or portion thereof, a gene, a nucleic acid sequence, a single-stranded DNA molecule ssDNA , a double-stranded DNA molecule dsDNA , an RNA molecule, an anti-sense RNA moiety, an siRNA moiety, an RNAi moiety, a double-stranded RNA moiety, a protein molecule or protein moiety, a protein antigen or portion thereof, an enzyme, a structural protein, a regulatory protein, a nutritional protein, a binding protein, a transport protein, a peptide molecule, a gene or genome of a fungus or portion thereof, or a synthetic cell.
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The biological product can also be a protein, peptide, or polypeptide that has undergone modification such as, for example, glycosylation of certain amino acid residues to produce a glycoprotein, or another molecule formed of two or more biological building blocks. In other embodiments the biological product is a genome of a synthetic bacteria or a portion thereof.
In a particular embodiment the biological product is an influenza virus and the sequence information is information that is used to prepare a vaccine against the biological threat. When the biological product is a virus or virus particle, it can be an attenuated virus or a killed virus or a harmless virus. In some embodiments the biological entity itself can also be the biological product. An example of when the biological entity is the biological product is a DNA vaccine, where the DNA molecule itself is useful as a vaccine.
After injection into the body the host cells synthesize the pathogen proteins, stimulating an immune response. In systems and methods where host cells are used they can be located within a subunit of the assembly unit that can receive a biological entity from another subunit for further processing, or can simply be maintained in a zone of the reaction container. For example the host cells can be maintained in a vessel within the subunit.
Host cells are useful in the systems and methods of the invention for a variety of purposes. While in vitro translation can be used in the systems and methods of the invention, transcription, translation, and assembly of molecular sub-units can also be performed by host cells. Host cells can also be used to receive a biological entity and use it to synthesize a biological product.
In one embodiment host cells are transfected with multiple DNAs, which are biological entities synthesized by a subunit of the assembly unit. Host cells may also be used to replicate phage or viral particles following infection or transfection. In the invention a digital DNA sequence or coded sequence can be entered into a software program that automatically designs a synthesis paradigm for the received DNA sequence e. The software can break the sequence into designed overlapping oligonucleotides of, for example, about bases or about bases or about bases or about bases or about bases or bases or bases or bases or bases or bases, and any of these sizes can be used as the oligonucleotides of the invention.
The oligonucleotide sequences are then transmitted to the sub-units of the system for assembly. The oligonucleotides can also be designed to have universal primer-binding domains for PCR amplification and restriction sites to release the primer-binding domains following the PCR amplification. The software used in the invention can also have the ability to modify received or desired sequence into codon-optimized sequences tailored to a particular host organism to be used in the method.
The software can be present in any unit or sub-unit of the system. In some embodiments the reaction container is a reaction plate. An example of a reaction plate is a 96 well plate, but it will be realized that the methods can be conducted on a reaction container having any number of reaction areas. When the reaction container is a reaction plate it can have any convenient number of reaction wells areas , such as the 96 reaction wells on a standard 96 well plate. These embodiments can involve dividing the reaction container into reaction zones, and transporting one or more samples from one zone of the reaction container to a second zone of the reaction container to perform distinct steps of a method, but can also involve leaving all or a portion of the sample in place and moving the reaction container so that one or more zones of the reaction container are exposed to a different environment.
For example, in a step involving PCR the zone can be located at a point in the system where it is exposed to a thermocycling schedule. At a DNA assembly step the reaction container can be located at another point in the system where it is exposed to the conditions appropriate for DNA assembly. The change of location can be accomplished by a physical movement of the reaction container or, alternatively, by a movement of one or more units of the system.
The reaction container is thus moved relative to the system of the invention. Normally each zone of a reaction container will contain multiple reaction areas, such as the wells in a reaction plate. But a reaction area does not require a physical barrier or boundary - it requires only that a distinct reaction can be carried out in the reaction area relative to other reaction areas.
A reaction container can have any number of zones but each zone has one or more reaction areas for collecting and holding a sample. Each zone of a reaction container is treated in the same manner or subjected to the same treatment or process in a given time period. For example the samples present in a zone may all be cycled at the same temperature of a PCR cycle being executed on that zone of the reaction container, or all reaction samples in a reaction zone may be picked up in automated fashion and moved to another zone on the same reaction container.
The methods can also have a step of DNA assembly performed in another reaction zone of the reaction container, and a transcription step performed in another reaction zone of the reaction container, and a translation step performed in another reaction zone of the reaction container, and a transfection step performed in another reaction zone of the reaction container. Thus, each step of any of the methods described herein can be performed in a distinct reaction zone of a reaction container.
The kits can contain reagents and components necessary to assemble a biological product, such as any combination of biological building blocks, building block polymers, buffers, or other reagents for carrying out a method of the invention on a system of the invention. Either alone or with any combination of the above reagents and components, the kits can also contain a reaction container for performing methods of the invention.
Introducing WYD Minute - SDM EN
Any combination of the reagents or reaction components or reaction containers can be provided in a container having the members of the kit. Reagents and reagent components can also be provided in a reagent vessel that fits onto an interface of a system of the invention. The instructions can also be provided on a website and the kit can include a link to the website, either instead of or in addition to the instructions provided with the kit. For example, the instructions can provide guidance on the type and quantity of reagents to be used in the systems and methods. The kits can be provided or packaged in a single container or in multiple containers.
The vessels can be made of glass, plastic or any suitable material, and may also be sterile. One or more vessels can be provided in a sterile container within the container comprising the kit. The biological entity can be any desired sequence. For example the systems of the invention can be pre-programmed to enable the operator to append a particular sequence to be synthesized to a plasmid or other vector desirable for the further use of the synthesized sequence.
In other embodiments the systems can synthesize a requested sequence and append regulatory sequences or promoter sequences or binding elements for trans-acting factors, or signal sequences to the requested sequence as part of the biological entity. Thus, when the operator knows that a sequence will be synthesized for later use in a particular host organism, the system can synthesize the requested sequences with the regulatory sequences or other desirable sequences particular to the host organism, thus further simplifying and speeding the preparation of the biological entity or biological product.
These pre-made sequences can also be included in any kit of the invention. The biosecurity screening is done by comparing the sequence the system has received in all 6 reading frames and is requested to synthesize against a pre-programmed database of prohibited sequences. The prohibited sequences can be derived from a list of pathogens, biological weapons, or other biosecurity threats that are prohibited by a local government from being synthesized.
The system can be programmed so that if a sequence is received that is identified on the list of prohibited sequences the system will be rendered inoperative or will otherwise not synthesis a prohibited sequence. Prohibited sequences can also be obtained from the International Gene Synthesis Consortium. The system can also screen against protein sequences derived from a prohibited sequences list.
In one embodiment the modification is the methylation of DNA at particular nucleotides or nucleotide analogs. In another embodiment the system produces a requested RNA molecule and adds a 7-methylguanosine residue to the 5 ' end as a 5 ' cap or a 5 '-5' phosphate linkage as a cap, which the system can then methylate to form mature mCAP. Guanosine-5'-triphosphate-5'guanosine can also be used as an RNA cap.
RNA bases can also be post-transcriptionally modified by the systems using a 2' O-methyl group to increase melting temperature and increase stability. In another embodiment the system produces a requested RNA molecule having a poly-A tail, with any number of A residues. Other embodiments include the inclusion of cleavage signals or sequences, or GU-rich sequences in the biological entity, which also can be tailored to a particular host cell to be used in a subsequent procedure. In still more embodiments the systems of the invention produce a requested peptide, poly-peptide or protein sequence with.
Influenza viruses are made of a viral envelope containing glycoproteins wrapped around a central core. The influenza genome typically contains eight pieces of RNA with each containing one or two genes encoding viral proteins. In the case of influenza A, the genome contains 11 genes on eight pieces of RNA, encoding for 11 proteins, including hemagglutinin HA and neuraminidase NA.
These glycoproteins have key functions in the life cycle of the virus, including assisting in binding to host cells and reproduction of viral particles. The assembled virus containing these proteins is therefore useful in the production of a vaccine. Oligonucleotide Synthesis and Assembly.
The HA and NA constructs were approximately 3 kb in length and were assembled from 96 oligonucleotides in the method. The first and last oligonucleotides contained primer binding domains for PCR amplification and Notl restriction sites to release the primer binding domains following amplification and expose overlapping regions for DNA assembly, if necessary to assemble larger fragments. Each process can be performed in a distinct reaction zone of the reaction container which is a 96 well plate , and the reaction zone can be one or more columns on the 96 well plate.
For each assembled product PCR reactions were performed in automated fashion:. Thermal-cycle occurred using the following parameters:. Nucleic acid constructs of approximately 3 kb were produced. The electrophoretic gels are shown in Fig. These genes already include promoter regions pol I and pol II for expression following transfection into mammalian cells. The following were combined:. The genes are then transfected into MDCK cells to produce a rescued virus.
The cells then produce the virus product, which is ready for harvest.
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Example 2 - Generation of Immunological Response. The integrity of the transcribed RNA was demonstrated by agarose gel electrophoresis not shown. The cells expressed the H7 HA, as demonstrated by gel electrophoresis and Western blot with an H7-specific antibody. In one embodiment the phage is a bacteriophage. Phage therapy is useful for the treatment of pathogenic bacterial infections, in humans, animals, and plants. Phage therapy can be particularly useful in applications where the bacteria does not respond to convention methods of control.
NC , sequence below at a laboratory. The software divides the sequence into four overlapping fragments of about 1. Each fragment overlaps the next by 40 bp to form a circular molecule when assembled. Each of the four fragments is further divided into overlapping oligonucleotides. These oligonucleotides are about 64 bases in length with 32 bp overlaps.
The first and last oligonucleotides of each of the four overlapping fragments contain primer binding domains for PCR amplification and Notl restriction sites to release the primer binding domains, following amplification, and expose overlapping regions for DNA assembly. Following entry the biological sequence information is transmitted to a receiving unit located in a laboratory in a remote city. All sub-units of the assembly unit are set up prior to receiving the biological sequence information.
Setup includes, but is not limited to, charging all vessels with requisite chemicals, reagents, and biological building blocks, as well as preparing all software programming prior to receiving the biological sequence information so that activation of the synthesis of the biological entity can begin immediately upon receiving the sequence information. Each step of the method is performed in a distinct reaction zone of the reaction container.
This synthesis is performed on a sub-unit such as an oligonucleotide synthesizer. Other packings can also be used that are stable in the pH range Thus, the ammonium hydroxide solution, diluted with water, is loaded directly onto the packing. After elution of failure sequences, the trityl protecting group is removed and washed from the support-bound oligonucleotide. The fully deprotected product can then be eluted and isolated by lyophilization.
Following the above procedure the four pools of oligonucleotides making up each of the four fragments required to produce overlapping dsDNA fragments of PhiX are produced. Each sub-unit of the assembly unit is pre-charged with reagents as necessary to perform all steps. After oligonucleotide assembly a robotic arm is used to transfer the reaction container from the oligo synthesizer to a liquid handler with thermo-cycling capabilities.
The protocol is set forth below. For each assembled product PCR reactions are performed in automated fashion:. The following four PCR reactions are also performed in automated fashion by the thermo-cycling sub-unit of the assembly unit:. These steps are also performed in automated fashion. In this embodiment, synthetic genomes are incubated for 2 hours in one of the cell-free expression kits described above.
This is prepared as described below. Six ml of this buffer can be prepared by combining the following:. This can be prepared by combining the following:. The enzymes remain active following at least 10 freeze-thaw cycles. The mixture is ideal for the assembly of DNA molecules with bp overlaps. The first and last oligonucleotides contain primer binding domains for PCR amplification and NotI restriction sites to release the primer binding domains following amplification and expose overlapping regions for DNA assembly, if necessary to assemble larger fragments.
Additional sub-units include an automated in vitro translation system containing vessels with reagents for carrying out a cell-free translation of nucleic acid into protein. Following entry into the transmitting unit the biological sequence information is transmitted to a receiving unit located in a laboratory in a remote city.
In this embodiment the receiving unit is a computer connected to the same computer network as the transmitting unit. The additional sub-units of the assembly unit, including the sub-unit for in vitro translation, are all set up prior to receiving the biological sequence information by charging all vessels with requisite chemicals, reagents, and biological building blocks, as well as preparing all software programming prior to receiving the biological sequence information so that activation of the synthesis of the biological entity can begin immediately upon receiving the sequence information.
This step is done in the same manner as described above in the Examples and the set of. Different kits are available depending on the type and size of protein to be translated. The reaction zone of the reaction container where the reaction is to be performed is pre-charged with all necessary reagents. Following the expression reaction, a black light is held to the reaction mixture and the presence of translated GFP is confirmed by the emission of green light.
The protein is encoded by the ERBB2 gene. A number of antibody therapies are available that target HER A vaccine can be made containing the HER-2 antigen, which can be administered to a patient who will then form an antigenic response to the vaccine and produce antibodies to HER The vaccine can be useful in cancer treatments, and in any therapy where the production of HER-2 antibodies finds useful application.
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Suitable regulatory sequences are included in the sequence. Each fragment is of appropriate length for gene synthesis and is designed to overlap the next fragment by an appropriate number of base pairs such as, for example, about 40 bp. The biological sequence information is transmitted by a transmitting unit to a receiving unit of a system of the invention. As explained herein the gene can be assembled as a series of overlapping oligonucleotides, and a sub-unit of the assembly unit assembles the.
Suitable regulatory sequences for expression are included. The assembled gene is transferred to another sub-unit of the assembly unit or to another zone of the reaction container and cell-free, in vitro transcription is performed, and then to another sub-unit or another zone for translation of the DNA sequence, as explained herein. A purification step is performed if desired. The harvested HER-2 may be formulated and is utilized as an antigenic vaccine. The DNA sequence encoding for one or more HIV proteins is entered into a software program that separates the DNA sequence into overlapping oligonucleotide fragments of appropriate length, including DNA sequences of an appropriate plasmid e.
As described herein, the receiving unit provides the sequence to an assembly unit of the invention and the assembly unit begins synthesis of overlapping oligonucleotides, which will be assembled by a subunit of the assembly unit or by the same sub-unit in a different zone of the reaction container into the full plasmid vaccine. When injected into the patient the DNA vaccine will provide instructions to cells to make one or more HIV proteins, which will then provoke an immune response.
The technique is applicable to any DNA virus that can be incorporated into a plasmid or other vector that can be injected into the patient to be treated to provide immunity. The present invention can be applied in the rapid production of sub-unit vaccines. The nucleic acid sequences are separated into fragments as described above using an appropriate software program, with appropriate regulatory sequences included.
After synthesis of the nucleic acid, as described above, the nucleic acid is transferred to a sub-unit of the assembly unit or to another zone of the reaction container for in vitro translation. The vaccine can be produced in a sub-unit where a cell culture is being maintained, which sub-unit has been previously prepared with an established culture of MDCK cells a canine kidney cell line in appropriate media. A vaccine against human papilloma virus HPV can be. These VLPs can be assembled in yeast, insect cells, mammalian cells, or bacteria.
Multiple HPV LI virus-like particles can be included in a single vaccine for the broadest spectrum of immunity. The nucleic acid sequences coding for these proteins are identified and separated into fragments as described above using an appropriate software with appropriate regulatory sequences for expression in the chosen cell type included. The nucleic acid sequence information is then transmitted by a transmitting unit to a receiving unit of the invention located in a remote location.
The receiving unit provides the sequence information to the assembly unit of the system, and the VLPs are synthesized. As described above, the DNA molecule can be assembled from overlapping oligonucleotide fragments into one or more whole dsDNA molecules. The vaccine is produced by the cells in that sub-unit.
The translated proteins are provided to another sub-unit of the system where they are pooled and self-assemble into VLPs that are useful as a vaccine against HPV. The influenza A genome is composed of eight viral gene segments, including the HA and NA segments, which are important in immune response to the virus. In one embodiment the assembly unit has a subunit maintaining, for example, MDCK cells, T cells, or Vera cells for virus production these components can also be maintained in one or more additional zones of the reaction container. Plasmids containing multiple transcription cassettes can be constructed containing the eight viral genes and necessary regulatory sequences.
The eight genes can be present on multiple plasmids or a single plasmid if desired. The HA and NA segments are based upon sequences derived from a virus that is presenting a local threat. Thus, in one embodiment the six non-varying genes are prepared ahead of time and only the HA and NA genes are synthesized by the system. The six non-varying genes can then be treated as reagents in the method.
But in another embodiment a subunit of the assembly unit synthesizes the plasmids based upon information received from the receiving unit. In either embodiment, after synthesis the plasmids are transfected into the cell culture being maintained in another subunit of the assembly unit under conditions for high transfection efficiency.
The transfected cells produce whole virus particles which can be formulated as a live, attenuated vaccine, or as a killed or further inactivated vaccine, as desired. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.
For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. Country of ref document: Kind code of ref document: Date of ref document: The present invention provides a system for receiving biological sequence information and activating the synthesis of a biological entity.
The system has a receiving unit for receiving a signal encoding biological sequence information transmitted from a transmitting unit. The transmitting unit can be present at a remote location from the receiving unit. The system also has an assembly unit connected to the receiving unit, and the assembly unit assembles the biological entity according to the biological sequence information. Thus, according to the present invention biological sequence information can be digitally transmitted to a remote location and the information converted into a biological entity, for example a protein useful as a vaccine, immediately upon being received by the receiving unit and without further human intervention after preparing the system for receipt of the information.
The invention is useful, for example, for rapidly responding to viral and other biological threats that are specific to a particular locale. It would be very desirable to have a system that allows the transmission of biological information in digital form across great distances, and then the conversion of that digital information into any of a wide variety of biological entities. Kellee marked it as to-read Feb 26, Rakella marked it as to-read May 19, Neyda marked it as to-read May 23, Jan marked it as to-read May 27, Micielle marked it as to-read Feb 05, Frederick Rotzien marked it as to-read Feb 05, Betty marked it as to-read Feb 05, Carla marked it as to-read Feb 05, Nicola Fantom marked it as to-read Feb 05, Pat marked it as to-read Feb 05, Claire marked it as to-read Feb 05, Stella Clarkson marked it as to-read Feb 05, Reader marked it as to-read Feb 05, Emiley Allen Bowes marked it as to-read Feb 05, Agnes marked it as to-read Feb 05, Marc Parijs marked it as to-read Feb 05, Sue marked it as to-read Feb 05, Dawn Obrien marked it as to-read Feb 05, LLL Reads marked it as to-read Feb 05, Ann Ellis marked it as to-read Feb 05, Susan marked it as to-read Feb 05, Stacia Chappell marked it as to-read Feb 05, Cheryl Bradley marked it as to-read Feb 05, Louise Carlson Stowell marked it as to-read Feb 05, Diane marked it as to-read Feb 05, Garrity marked it as to-read Feb 05, Pam Mooney marked it as to-read Feb 05, D marked it as to-read Feb 05, Roxanne marked it as to-read Feb 05, The Nimnad - 6 copies to giveaway 1 8 Feb 07, The Nimnad - Goodreads Giveaway 1 4 Feb 05, Trivia About The Nimnad.
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