KAPLAN_USMLE_STEP_1_LECTURE_NOTES_2018_BIOCHEMISTRY_and_GENETICS

Part I ● Biochemistry

Recombinant Proteins

High-Yield

Recombinant proteins can be made by cloning the relevant MEDIUMgene for theYIELDprotein in a host organism, growing large quantities of the organism, and inducing it to express the gene (as indicated in the lower right of Figure I-6-1). Many therapeutics proteins are now mass-produced as recombinant proteins.

Table I-6-2. Examples of Protein Products of Recombinant DNA Technology

	Product			Produced in			Use
Insulin				E. coli			Diabetes
Growth factor				E. coli			Growth defects
Epidermal growth factor				E. coli			Burns, ulcers
Hepatitis B vaccine				Saccharomyces			Prevention of viral
				cerevisae			hepatitis
Erythropoietin				Mammalian cells			Anemia
Factor VIII				Mammalian cells			Hemophilia

Note

•Ex vivo: cells modified outside the body, then transplanted back in

•In vivo: gene changed in cells still in body

Gene Therapy	High-Yield

Gene therapy now offers potential cures for individuals withMEDIUMinheritedYIELDdiseases. The initial goal is to introduce a normal copy of the gene that is defective into the tissues that give rise to the pathology of the genetic disease.

For instance, about 50% of children with severe combined immunodeficiency have a mutation in the gene encoding the γ chain common to several of the interleukin receptors. Recently, cDNA from a normal γ-chain gene was used to transduce autologous cells from infants with X-linked severe combined immunodeficiency (SCID) with subsequent correction of the defects in their T cells and natural killer cells.

•Gene transfer requires a delivery vector (retrovirus, adenovirus, liposome).

•Only tissues giving rise to the disease pathology are targeted for gene therapy.

•The normal gene is not inherited by offspring.

Gene delivery vectors

For gene replacement therapy to be a realistic possibility, efficient gene delivery vectors must be used to transfer the cloned gene into the target cells’ DNA. Because viruses naturally infect cells to insert their own genetic material, most gene delivery vectors now in use are modified viruses. A portion of the viral genome is replaced with the cloned gene (as either DNA or RNA) such that the virus can infect but not complete its replication cycle. Steps in the production of a retrovirus for gene replacement therapy are illustrated in Figure I-6-7.

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Retroviruses and adenoviruses were early vectors used in gene delivery. However, newer strategies take advantage of vectors with better properties, such as adeno-associated viruses (AAV). Major advantages of AAV include having no disease association in humans and limited innate immunity. In addition, AAV restricts expression to specific tissues: A tissue-specific promoter in the AAV is genetically engineered to control transcription of the inserted transgene.

Table I-6-3. Vectors Used in Gene Therapy

										Adeno-Associated
	Viral Vector			Retroviruses			Adenovirus
										Virus (AAV)
	Family			Retroviridae			Adenoviridae			Parvoviridae
	Genome			ssRNA			dsDNA			ssDNA
	Disease			Yes			Yes			No
	association?
	Inserts into			Yes			No			No (episomal)
	chromosome?
	Innate immunity?			Yes			Yes			Limited

Retrovirus

Therapeutic human gene

Retroviral genes are replaced with therapeutic human gene, making the retrovirus incapable of self-replication

Package modified retroviral genome in host packaging cell

Multiple virions, carrying the retrovirus, are produced

Virions

Figure I-6-7. Preparation of a Retrovirus for Gene Replacement Therapy

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Part I ● Biochemistry

There are 2 strategies for delivering a therapeutic gene (transgene) into an individual.

•In vivo gene replacement therapy involves the direct delivery of a therapeutic gene into a patient’s body. Upon entry into the target cells, the inserted transgene is expressed into a therapeutic protein.

•Ex vivo gene replacement therapy involves the genetic manipulation of a patient’s target cells outside the body. Target cells are infected with a recombinant virus harboring the therapeutic transgene. The genetically modified target cells, harboring and expressing the therapeutic protein, are then reintroduced into the same patient.

	Cloned therapeutic transgene
In Vivo Therapy	inserted into viral delivery	Ex Vivo Therapy
(Direct Delivery)	vehicle	(Cell-Based Delivery)

Target cells infected	Target organ	Cells to be
	(e.g., liver)	genetically modified
with recombinant		(e.g., stem cells)
virus and integrated
into host chromosome

Target cells modified by transgene-containing

virus

Genetically modified cells into patient

Figure I-6-8. In Vivo and Ex Vivo Gene Replacement Therapies

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Chapter 6 ● Genetic Strategies in Therapeutics

Gene replacement therapy (in vivo therapy) for cystic fibrosis illustrates an important example of direct delivery of a transgene.

Cloned therapeutic transgene inserted into viral delivery vehicle (AAV)

Bacterial cloning	AAV-containing
of human	therapeutic
therapeutic gene	transgene nasally
	dripped into lung

AAV-containing therapeutic transgene enters lung cell nucleus

Nucleus

Lung cell

Therapeutic transgene integrated into lung cell chromosomes

Therapeutic transgene corrects genetic defect

Figure I-6-9. Gene Replacement Therapy for Cystic Fibrosis

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Part I ● Biochemistry

An important example of ex vivo gene replacement therapy is illustrated below.

Retrovirus with cloned

IL-R γ-chain gene

CD34+ bone marrow cells

Culture with growth factors

Infuse modified cells into patient

Figure I-6-10. Ex Vivo Gene Replacement Therapy for X-Linked Severe Combined Immunodeficiency

Remaining challenges to gene replacement therapy

Although much progress has been made in gene replacement therapy, significant challenges still remain. These challenges include:

•Targeting the therapeutic gene to the appropriate tissues

•Low-level or transient expression of the therapeutic gene

•Problems caused by random insertion of the therapeutic gene into the host DNA.

Recall Question

Which of the following is true for both genomic libraries and cDNA (expression) libraries?

A.Cloned genes contain introns

B.DNA ligase is used

C.Reverse transcriptase is used

D.Can be used for gene therapy

Answer: B

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Chapter 6 ● Genetic Strategies in Therapeutics

RNA Interference

RNA interference (RNAi) refers to downregulation of gene expression through the use of small RNA molecules, which mediate gene expression by either inhibiting translation or causing premature degradation of the genes’ mRNAs. Two types of small RNA molecules are involved in RNA interference: microRNA (miRNA) and small interfering RNA (siRNA). The RNAi pathway occurs in many eukaryotes, including humans, and plays a central role in defending cells against viruses and transposons (discussed in Microbiology Lecture Notes).

The RNAi process begins when an enzyme known as dicer cleaves long doublestranded RNA into small double-stranded RNA fragments (siRNA) approximately 20 nucleotides in length. The double-stranded siRNA then unwinds into two single-stranded RNAs: the passenger strand (sense strand), which subsequently degrades, and the guide strand (antisense strand), which associates into the RNA-induced silencing complex (RISC). Next, the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage using argonaute, the catalytic component of the RISC complex. Since the mRNA is degraded, its encoded protein is not produced. The result is posttranscriptional gene silencing. This effect is often referred to as “knockdown” because gene expression continues, though in greatly reduced extent. In “knockout,” by contrast, gene expression is entirely absent.

dsRNA

Dicer

siRNA

Attachment of RISC to the “anti-sense” RNA strand

Degradation of the “sense” RNA strand

mRNA

Degradation of mRNA

Figure I-6-11. RNAi Pathway

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To time-limit the effects of RNAi, siRNAs can be administered as stabilized RNAs conjugated to targeting compounds or enclosed in lipid vesicles. A single RNAi treatment can silence expression of a particular gene for 2 weeks. siRNAs can be stabilized against endogenous RNases in blood or cells by modifying the 2' hydroxyl with a methyl or fluorine group.

RNA interference technology is being explored as a treatment for cancer and many neurodegenerative diseases and for use in antiviral therapies. Clinical trials are also exploring the use of RNAi in other clinical applications, such as therapy for age-related macular degeneration.

Table I-6-4. Summary of Important Points About Recombinant DNA

	Restriction	Recognize palindromes in dsDNA:
	endonucleases		5′ - - - G A A T T C - - - 3′
			3′ - - - C T T A A G - - - 5′
		Cut leaving sticky ends:
			5′ - - - G A A T T C - - - 3′
			3′ - - - C T T A A G - - - 5′
		Used to make restriction maps of DNA
		Produce fragments for genetic analysis
		Produce fragments for making recombinant DNA and
		cloning DNA sequences
	Vectors for	Plasmid:
	recombinant	●●	Restriction site
	DNA and cloning
			Replication origin
		●●
		●●	Resistance to antibiotic(s)
		Expression vector also requires:
		●●	Promoter
		●●	Shine-Dalgarno sequence
		Other vectors: phage, YACs
	Approaches to	Genomic DNA
	cloning DNA	●●	Restriction endonucleases fragment DNA
		●●	Total nuclear DNA cloned
		●●	Genes contain introns
		cDNA
		●●	Reverse transcription of mRNAs from cell
		●●	Genes expressed cloned
		●●	Genes have no introns
	Uses of cloned	Produce recombinant proteins
	genes	Gene therapy (somatic)
		Transgenic animals (germline)
		Produce cDNA probes for blots

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Review Questions

1.If a patient with cystic fibrosis were to be treated by gene therapy, which type of cells should be targeted as host cells?

A.Germ cells

B.Epithelial cells

C.T cells

D.Hemopoietic stem cells

2.A pharmaceutical firm is interested in the bacterial production of thymidylate synthase in large quantities for drug-targeting studies. An important step in the overall cloning strategy involves the ligation of synthase cDNA into a plasmid vector containing a replication origin, an antibiotic resistance gene, and a promoter sequence. Which additional nucleotide sequence should be included in this vector to ensure optimal production of the thymidylate synthase?

A.Operator sequence

B.PolyA sequence

C.Shine-Dalgarno sequence

D.Attenuator sequence

E.3′-splice acceptor sequence

3.Restriction fragment length polymorphisms may be produced by mutations in the sites for restriction endonucleases. For instance, a single base change in the site for the nuclear SalI produces the sequence GTGGAC, which can no longer be recognized by the enzyme. What was the original sequence recognized by SalI?

A.GTAGAC

B.GCGGAC

C.CTGGAC

D.GTCGAC

E.GTGTAC

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Answers

1.Answer: B. The pathogenesis of cystic fibrosis is related to defective chloride transport in epithelial cells.

2.Answer: C. Incorporation of a Shine-Dalgarno sequence into the expression vector will promote ribosome binding to the translation start site on the mRNA produced by transcription of the cDNA insert.

3.Answer: D. All options represent single-base changes in the mutant sequence in the stem, but only choice D reestablishes a palindrome.

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Techniques of Genetic Analysis

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Learning Objectives

Interpret scenarios about blotting technique

Explain information related to polymerase chain reaction

Techniques of genetic analysis are assuming an increasingly larger role in medical diagnosis. These techniques, which once were a specialized part of medical genetics, are now becoming essential tools for every physician to understand. Blotting techniques allow testing for genetic diseases, gene expression profiling, and routine testing for antigens and antibodies. The polymerase chain reaction (PCR) is now an essential tool in many aspects of genetic testing, forensic medicine, and paternity testing. These techniques are discussed in this chapter, but their applications will be further explored in Medical Genetics (Section II of this book).

BLOTTING TECHNIQUES

Blotting techniques have been developed to detect and visualize specific DNA, RNA, and protein among complex mixtures of contaminating molecules. These techniques have allowed the identification and characterization of the genes involved in numerous inherited diseases.

Add probe

Visualize

Transfer to

to reveal

bands

bands of

(autoradio-

membrane

interest

graphy)

Material separated

Material on blot

Solid lines

by gel electrophoresis

represent bands

reactive with probe

Figure I-7-1. Blotting Technique

The fragments in the material to be analyzed (DNA, RNA, or protein) are separated by gel electrophoresis. The smaller molecules travel faster and appear nearer the bottom of the gel. The bands of material in the gel are transferred, or blotted, to the surface of a membrane. The membrane is incubated with a (usually radioactive) labeled probe that will specifically bind to the molecules of interest. Visualization of the labeled probe (usually by autoradiography) will reveal which band(s) interacted with the probe.

Only the bands reactive with probes are made visible

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