3686 PART 16 Genes, the Environment, and Disease

■ IMMUNODEFICIENCY DISORDERS: PROOF OF

PRINCIPLE FOR EX VIVO GENE TRANSFER

Early attempts to effect gene replacement into hematopoietic stem cells

(HSCs) were stymied by the relatively low transduction efficiency of

retroviral vectors, which require dividing target cells for integration.

Because HSCs are normally quiescent, they are a formidable transduction target. However, identification of cytokines that induced cell

division without promoting differentiation of stem cells, along with

technical improvements in the isolation and transduction of HSCs, led

to modest but real gains in transduction efficiency.

The first convincing therapeutic effect from gene transfer occurred

with X-linked severe combined immunodeficiency disease (SCID),

which results from mutations in the gene (IL2RG) encoding the γc subunit of cytokine receptors required for normal development of T and

natural killer (NK) cells (Chap. 351). Affected infants present in the

first few months of life with overwhelming infections and/or failure to

thrive. In this disorder, it was recognized that successfully transduced

cells, even if few in number, would have a proliferative advantage compared to nontransduced cells, which lack receptors for the cytokines

required for lymphocyte development and maturation. Isolation of

autologous CD34+ cells, followed by transduction with a retroviral vector encoding the γc subunit and transplantation of the gene-modified

autologous cells, led to complete reconstitution of the immune system,

including documented responses to standard childhood vaccinations,

clearing of infections, and remarkable gains in growth in most of the

treated children. However, among 20 children treated in the initial

trials, five eventually developed a syndrome similar to T-cell acute

lymphocytic leukemia, with splenomegaly, rising white blood cell

counts, and the emergence of a single clone of T cells. Molecular studies revealed that, in most of these children, the retroviral vector had

integrated within a gene, LMO-2 (LIM only-2), that encodes a component of a transcription factor complex involved in hematopoietic

development. The retroviral long terminal repeat acted as a promoter

to increase the expression of LMO-2, resulting in T-cell leukemia.

The X-linked SCID studies were a watershed event in the evolution

of gene therapy. They demonstrated conclusively that gene therapy

could cure disease, with dramatic and durable clinical results. However,

they also demonstrated that insertional mutagenesis leading to cancer

was more than a theoretical possibility (Table 470-4). As a result of

the experience in these trials, all protocols using integrating vectors in

hematopoietic cells must include a plan for monitoring sites of insertion and clonal proliferation for 15 years after infusion (although to

date, insertional mutagenesis has never been reported in over several

thousand patient-years of experience where the transduced cell is a

mature T cell rather than an HSC). Strategies to overcome this complication have included using a “suicide” gene cassette in the vector, so

that errant clones can be quickly ablated, or using “insulator” elements

in the cassette, which can limit the activation of genes surrounding

the insertion site. The occurrence of malignancy in the X-linked SCID

trials has fueled a transition to lentiviral vectors. These efficiently

TABLE 470-2 Currently Approved Gene and Cell Therapy Products in the United States and/or Europe

PRODUCT INDICATION AGE GROUP

YEAR FIRST

APPROVED

WHERE

APPROVED VECTOR TRANSGENE TARGET TISSUE

Strimvelis®a ADA-SCID Pediatric 2016 Europe Retroviral ADA (adenosine

deaminase)

Autologous

hematopoietic stem

cells (HSCs)

Kymriah®

Tisagenlecleucel

Relapsed or refractory (R/R)

B-cell acute lymphoblastic

leukemia (pediatric); R/R large

B-cell lymphoma (adult)

Pediatric and

adult, different

disease indications

2017 U.S. and Europe Lentiviral Chimeric antigen

receptor with

4-1BB signaling

domain

Autologous T cells

Yescarta®

Axicabtagene

ciloleucel

R/R large B-cell lymphomas Adult 2017 U.S. and Europe Retroviral Chimeric antigen

receptor with CD28

signaling domain

Autologous T cells

Luxturna®

Voretigene

neparvovec

Confirmed biallelic RPE65

mutation-associated retinal

dystrophy

Pediatric and adult 2017 U.S. and Europe AAV RPE65 (retinal

pigment epithelial

65-kDa protein)

Retinal pigment

epithelial cells

Zolgensma®

Onasemnogene

abeparvovec

Spinal muscular atrophy type

1 due to biallelic mutations in

the SMN1 gene

Pediatric <2 years

of age

2019 U.S. and Europe AAV SMN1 (survival

motor neuron 1)

Spinal motor

neurons

Zynteglo®

Betibeglogene

autotemcel

Transfusion-dependent β

thalassemia

Adult and pediatric

≥12 years of age

2019 Europe Lentiviral bA-T87Q-globin gene Autologous HSCs

Libmeldy®b Metachromatic

leukodystrophy due to biallelic

mutations in the arylsulfatase

A gene

Pediatric 2020 Europe Lentiviral ARSA

(arylsulfatase A)

Autologous HSCs

Tecartus®

Brexucabtagene

autoleucel

R/R mantle cell lymphoma Adult 2020 U.S. Retroviral Chimeric antigen

receptor with CD28

signaling

Autologous T cells

a

Autologous CD34+-enriched cell fraction that contains CD34+ cells transduced with retroviral vector that encodes for the human ADA cDNA sequence. b

Autologous CD34+

cells encoding arylsulfatase A.

Abbreviations: AAV, adeno-associated virus; ADA-SCID, adenosine deaminase severe combined immunodeficiency.

TABLE 470-3 Most Common Indications in Gene Therapy Trials

INDICATION NUMBER

Cancer 1688

Monogenic diseases 287

Infectious diseases 182

Source: Adapted from SL Ginn et al: Gene therapy clinical trials worldwide to 2017:

An update. J Gene Med 20:e3015, 2018.

TABLE 470-4 Potential Complications of Gene Therapy

Gene silencing—repression of promoter

Genotoxicity—complications arising from insertional mutagenesis

Phenotoxicity—complications arising from overexpression or ectopic expression

of the transgene

Immunotoxicity—harmful immune response to either the vector or transgene; or

a harmful immune response of the vector (e.g., CAR T cells)

Risks of horizontal transmission—shedding of infectious vector into environment

Risks of vertical transmission—germline transmission of donated DNA

Abbreviation: CAR, chimeric antigen receptor.

Gene- and Cell-Based Therapy in Clinical Medicine

3687CHAPTER 470

transduce nondividing target cells and are characterized by a different

pattern of integration into the genome that appears to be safer than

retroviral vectors. To date, there have been no reports of tumorigenesis

due to insertional events with lentiviral vectors; the field is thus gradually moving toward these to replace retroviral vectors for transduction

of cells other than T cells.

Gene therapy also produced clear success for another form of SCID,

that due to adenosine deaminase (ADA) deficiency (Chap. 351).

ADA-SCID is clinically similar to X-linked SCID, although it can be

treated by enzyme replacement therapy with a pegylated form of the

enzyme (PEG-ADA), which leads to immune reconstitution but not

always to normal T-cell counts. Enzyme replacement therapy is expensive (annual cost: $200,000–$300,000 [U.S. dollars]), and some patients

develop antibodies to PEG-ADA, reducing therapeutic efficacy. The

initial trials of gene therapy for ADA-SCID were unsuccessful, but

modifications of this protocol to include the use of HSCs rather than

T cells as the target for transduction; discontinuation of PEG-ADA

at the time of vector infusion, so that the transduced cells have a

proliferative advantage over the nontransduced cells; and the use of a

mild conditioning regimen to facilitate engraftment of the transduced

autologous cells led to success without the complications seen in the

X-linked SCID trials. This therapy (Strimvelis®) was approved in 2016

by the European Medicines Agency (Table 470-2). Recently, however,

a patient who received the treatment in 2016 has been diagnosed with

T-cell leukemia, and preliminary findings suggest an insertional event

related to the gene therapy as the cause. Thus, although the risk of

insertional mutagenesis appears to be much lower in the setting of

ADA-SCID (1 patient out of 36 treated in the clinical trials or with

the approved product), it is not absent. Despite this, ADA-SCID is

an example where gene therapy has changed therapeutic options for

patients. For those with a human leukocyte antigen (HLA)-identical

sibling, bone marrow transplantation is still the best treatment option,

but this includes only a minority of those affected. For those without

an HLA-identical match, gene therapy has comparable efficacy to PEGADA, does not require repetitive injections, and does not present the

risk of development of neutralizing antibodies to the bovine enzyme.

Continued observation of previously treated patients may change the risk/

benefit assessment and perhaps promote development of lentivirus-based

vectors for the disease.

■ TRANSFUSION-DEPENDENT THALASSEMIA:

EXTENSION OF PRINCIPLE

A goal of gene therapy is to provide therapeutic options for more

prevalent diseases such as thalassemia and sickle cell disease. These

red cell disorders are more challenging targets for gene therapy than

the immunodeficiencies for several reasons: first, in immunodeficiency disorders, the transduced cells have a survival advantage over

nontransduced cells, which is not the case in red cell disorders. Second, in order to achieve transfusion independence or freedom from

vaso-occlusive crises, one must achieve higher transduction efficiency

and engraftment of higher numbers of stem cells. Promising clinical

data exist now for both of these conditions, and the first product for

transfusion-dependent β thalassemia has been conditionally approved

in Europe (Table 470-2). Standard of care for transfusion-dependent β thalassemia (TDT) consists of lifelong regular red blood cell

transfusions, typically monthly, to support hemoglobin levels >9 g/

dL, coupled with an intensive regimen of iron chelation to minimize

iron overload to the liver, heart, and endocrine system (Chap. 98).

Allogeneic stem cell transplantation addresses the underlying cause

of the disease but carries risks of myeloablation, graft-versus-host

disease (GVHD), and graft rejection and thus is reserved primarily

for those with an HLA-matched sibling donor (<25% of patients). The

first approved gene therapy for β thalassemia consists of a lentiviral

vector driving expression of an antisickling variant of β-globin (the

same product is being developed for sickle cell disease), introduced

into autologous HSCs, which are then transplanted back into the

patient after myeloablation. Results of clinical trials showed that

15 of 19 patients with TDT due to genotypes other than β0

/β0

 (the

most severe genotype of the disease) achieved durable transfusion

independence that was maintained through follow-up ranging up to

5 years. The remaining four patients had substantial reduction in the

transfusion requirement. Gene therapy with lentiviral transduction of

autologous cells thus dramatically simplifies the medical regimen for

these patients, since it eliminates the need for transfusion and iron

chelation and carries no risk of GVHD or graft rejection because it is

generated from the patient’s own cells. Similarly, since the transduced

cells are autologous, there is no requirement for an HLA-matched

donor, expanding the numbers of patients who can be treated. Safety

in the initial trials has been excellent, with most adverse events related

to the known risks of the myeloablative conditioning regimen. Studies

currently underway aim to achieve the higher levels of transduction,

expression, and engraftment needed for patients with β0

/β0

 thalassemia

and those with sickle cell disease.

Encouraging results using a gene-editing approach provide another

therapeutic option for these patients. In recent trials, a CRISPR/Cas9

vector targeting the BCL11A gene, which normally represses γ-globin

(the fetal β-like globin), was introduced into autologous CD34+ cells of

patients with TDT or sickle cell disease. Results with up to 18 months

of follow-up show a gradual rise in fetal hemoglobin in the circulation

to levels that obviate the need for transfusion and, in sickle cell disease, the cessation of vaso-occlusive crises. The gene-editing approach

still requires myeloablation to ensure engraftment of the gene-edited

autologous cells. Studies designed to support licensing are underway;

currently, there are no approved gene-editing products.

■ NEURODEGENERATIVE DISEASES: BROADENING

OF PRINCIPLE

The SCID trials gave support to the hypothesis that gene transfer into

HSCs could be used to treat any disease for which allogeneic bone

marrow transplantation was therapeutic. Moreover, the use of genetically modified autologous cells carried the advantages noted above,

i.e., no risk of GVHD, guaranteed availability of a “donor” (unless the

disease itself damages the stem cell population of the patient), and

low likelihood of failure of engraftment. Investigators in Paris capitalized on this realization to conduct the first trial of lentiviral vector

transduction of HSCs for a neurodegenerative disorder, X-linked

adrenoleukodystrophy (ALD). The key to the mechanism of action is

that a subpopulation of the gene-modified cells give rise to myeloid

cells that cross the blood-brain barrier and engraft as central nervous

system (CNS)–resident microglia and perivascular CNS macrophages.

The transduced cells carry the gene encoding the missing protein, in

this case an adenosine triphosphate–binding cassette transporter. Following lentiviral transduction of autologous HSCs in young boys with

the disease, dramatic stabilization of disease occurred, demonstrating

that stem cell transduction could work for neurodegenerative as well as

immunologic disorders.

Investigators in Milan carried this observation one step further

to develop a treatment for another neurodegenerative disorder that

had previously responded poorly to bone marrow transplantation.

Metachromatic leukodystrophy is a lysosomal storage disorder caused

by mutations in the gene encoding arylsulfatase A (ARSA). The late

infantile form of the disease is characterized by progressive motor

and cognitive impairment and death within a few years of onset, due

to accumulation of the ARSA substrate sulfatide in oligodendrocytes,

microglia, and some neurons. Recognizing that endogenous levels

of production of ARSA were too low to provide cross-correction

by allogeneic transplant, a lentiviral vector was engineered to direct

supraphysiologic levels of ARSA expression in transduced cells. Transduction of autologous HSCs from children born with the disease, at

a point when they were still presymptomatic, has led to preservation

and continued acquisition of motor and cognitive milestones at time

periods as long as 8 years after treatment, with observation ongoing.

This product is approved in Europe for those with late infantile or

early juvenile forms of the disease (Table 470-2). These results illustrate

that the ability to engineer levels of expression can allow gene therapy

approaches to succeed where allogeneic bone marrow transplantation

cannot. It is likely that a similar approach will be used in other neurodegenerative conditions.

3688 PART 16 Genes, the Environment, and Disease

LONG-TERM EXPRESSION IN GENETIC

DISEASE: IN VIVO GENE TRANSFER WITH

RECOMBINANT ADENO-ASSOCIATED

VIRAL VECTORS

Recombinant adeno-associated viral (AAV) vectors have emerged as

attractive gene delivery vehicles for genetic disease. Engineered from a

small replication-defective DNA virus, they are devoid of viral coding

sequences and trigger very little immune response in experimental animals. They are capable of transducing nondividing target cells, and the

donated DNA is stabilized primarily in an episomal form, thus minimizing risks arising from insertional mutagenesis. Because the vector

has a tropism for certain long-lived cell types, such as skeletal muscle,

the CNS, and hepatocytes, long-term expression can be achieved even

in the absence of integration. Of note, because the donated DNA is

predominantly nonintegrated, long-term expression requires targeting

of nondividing or slowly dividing cells; otherwise, expression is lost as

cells divide.

■ FIRST LICENSED PRODUCT

These features of AAV were used to develop the first approved gene

therapy product in the Western world, an AAV vector conditionally

approved in Europe in 2012 for treatment of the autosomal recessive

disorder lipoprotein lipase (LPL) deficiency. This rare disorder (1–2

cases/million) is due to loss-of-function mutations in the gene encoding LPL, an enzyme normally produced in skeletal muscle and required

for the catabolism of triglyceride-rich lipoproteins and chylomicrons.

Affected individuals have lipemic serum and may have eruptive xanthomas, hepatosplenomegaly, and in some cases, recurrent bouts of

acute pancreatitis, and do not respond to treatment with statins. Clinical trials demonstrated the safety of intramuscular injection of AAVLPL and provided preliminary evidence supporting a reduction in

frequency of episodes of pancreatitis. The sponsor allowed the approval

to expire in 2017, without completing the postmarketing requirements,

but the initial approval, demonstrating a regulatory pathway for this

class of therapeutics, was a crucial catalyst for the current robust

activity in gene therapy, with ongoing trials in a range of diseases

including muscular dystrophies, Parkinson’s disease, Huntington’s disease, Pompe disease, hemophilia B and A, several forms of congenital

blindness, and a variety of other inherited conditions. Currently there

are two approved AAV gene therapies for genetic disease, one for a rare

form of congenital blindness and the other for the fatal pediatric neurodegenerative disease spinal muscular atrophy type 1 (Table 470-2).

■ RETINAL GENE THERAPY

The retina is an attractive target for AAV-mediated gene transfer: It

is a relatively immuneprivileged space, obviating problems related to

immune responses, and the photoreceptors, retinal ganglion cells, and

retinal pigment epithelial cells are long-lived postmitotic cells. Routes

of administration for these cell types—either intravitreal or by subretinal injection—involve standard procedures in ophthalmology. Given

the small space, doses required are relatively low, lessening the manufacturing burden. Finally, canine models of a number of inherited retinal dystrophies have been well characterized and faithfully reflect the

human disease. Work carried out in the 1990s had demonstrated that

the canine disease could be reversed, with durable restoration of vision,

by subretinal injection of an AAV vector in dogs with a mutation in the

gene encoding RPE65 (retinal pigment epithelial-associated 65-kDa

protein), an enzyme key to the visual cycle. Like the canine disease, the

human disease is characterized by early-onset visual impairment, with

most patients progressing to blindness over time. Phase 1 clinical trials

by multiple groups established the safety of subretinal injections of an

AAV vector expressing RPE65. A phase 3 trial, the first randomized

controlled trial in human gene therapy, demonstrated improvement in

multiple measures of retinal and visual function. Of note, and likely to

be a recurring theme as gene therapies address diseases for which there

are no existing treatments, successful clinical development required

the development and validation of a novel clinical endpoint, a mobility

test conducted at varying levels of environmental illumination, that

could measure improvements in functional vision. This product, the

first licensed AAV gene therapy product in the United States, is now

approved in both the United States and Europe (Table 470-2). Trials for

other inherited retinal degenerative disorders such as choroideremia

are underway, as are studies for certain complex acquired disorders

such as age-related macular degeneration, which affects several million

people worldwide. The neovascularization that occurs in age-related

macular degeneration can be inhibited by expression of vascular endothelial growth factor (VEGF) inhibitors such as angiostatin or through

the use of RNA interference (RNAi)–mediated knockdown of VEGF.

Early-phase trials of an AAV vector designed to achieve long-term

inhibition of the biologic effects of VEGF through a soluble VEGF

receptor, however, failed to provide convincing evidence of efficacy,

illustrating the challenges of developing genetic approaches for complex acquired disorders.

■ SPINAL MUSCULAR ATROPHY TYPE 1

Spinal muscular atrophy type 1 is the most common genetic cause of

death in infancy and affects ~1 in 11,000 births. The disease is caused

by autosomal recessive mutations in the SMN1 gene, encoding survival motor neuron 1; affected infants undergo degeneration and loss

of lower motor neurons, presenting as hypotonia, severe weakness,

and failure to sit without support. In a large natural history study

of untreated infants with the disease, by age 20 months, only 8% of

patients with the disease were alive and free of ventilator support. In

a phase 1 gene therapy study, intravenous infusion of an AAV vector

(one with tropism for the nervous system [AAV9]) expressing SMN1

showed survival without ventilator support in 100% of participants

(n = 15) at 20 months of age. A phase 3 trial was initiated, but the

treatment was approved in the United States based on the efficacy

data from the first 21 participants enrolled in that study, coupled with

the safety data from the ongoing phase 3 study and the completed

phase 1 study (Table 470-2). The major safety concern was the risk of

acute serious liver injury; because the vector is infused intravenously,

there is considerable biodistribution to the liver (and to the spinal motor

neurons, the therapeutic target). The approved dose of 1.1 × 1014 vg/kg

is quite high and results in marked elevation of liver transaminases

if untreated. The phase 1 study showed that this could be controlled

using a course of corticosteroids begun 1 day before the vector infusion

and continued for 30 days, with tapering at that point and carried out

with monitoring of liver transaminases. Patients receiving the approved

treatment are followed in a registry designed to assess effectiveness, longterm safety, and overall survival of patients with spinal muscular atrophy.

An alternative treatment, intrathecal administration of an antisense

oligonucleotide designed to increase expression of functional SMN from

a mostly nonfunctional SMN2 gene, was approved during the course of

the gene therapy studies, but the efficacy data in the clinical trial were less

robust, and the drug requires intrathecal administration every 4 months.

Clearly, the gene therapy approach is a disease-modifying one, for a

disease that previously lacked treatment. Additional follow-up in this

population should address questions of long-term safety and efficacy.

GENE THERAPY FOR CANCER

The majority of clinical gene transfer experience has been in subjects

with cancer. The intent has been to increase the precision of cancer

therapies and thereby make them less toxic and more effective. Most

approaches have either modified the tumor directly or altered the host’s

response to the malignancy to produce immune effector cells that are

precisely targeted to the tumor phenotype.

■ MODIFYING THE CANCER

Since cancer is an (acquired) genetic disorder, initial efforts were

directed at correcting the genetic deficits of the tumor or introducing

lethal genes. Two major and persisting obstacles, however, are the poor

biodistribution and transduction efficiency of all currently available

vectors and the heterogeneity and genetic instability of the tumor targets themselves, so that correction of single driver mutations does not

preclude the evolution of a resistant population.

Tumor Correction One widely used direct intratumoral approach

was adenoviral-mediated expression of the tumor suppressor p53,

Gene- and Cell-Based Therapy in Clinical Medicine

3689CHAPTER 470

which is mutated in many different cancers. Initial studies showed

some complete and partial responses in squamous cell carcinoma of the

head and neck, esophageal cancer, and non-small-cell lung cancer, but

as yet, there have been no successful product licensing studies for this

approach except in China.

Prodrug Metabolizing Genes Efforts to overcome the above

limitations have included the introduction of a prodrug or a suicide

gene that would increase sensitivity of tumor cells to cytotoxic drugs.

An early strategy was intratumoral injection of an adenoviral vector

expressing the thymidine kinase (TK) gene. Cells that take up and

express the TK gene can be killed after the administration of ganciclovir, which is phosphorylated to a toxic nucleoside by TK. The advantage of this approach is that the effects of transducing even a limited

number of tumor cells are amplified by the spread of active drug to

adjacent tumor cells. Although the approach continues to be examined

in aggressive brain tumors and locally recurrent prostate, breast, and

colon tumors, progress remains slow, and systemic benefits against

metastatic disease have not been established.

■ MODIFYING THE HOST

Recruiting the Immune System The successful use of monoclonal antibodies that produce antitumor activity by activating the

immune response has demonstrated the feasibility of manipulating the

immune system to recognize the abnormal pattern of antigen expression on tumor cells. Immune cells are capable of almost unlimited

expansion and persistence and can provide long-term tumor control.

They can also traffic to tumor sites irrespective of location and, in principle, have the potential to evolve with the changing pattern of tumor

cell phenotype and function. Antibodies targeting “checkpoint” molecules, particularly CTLA-4 and the PD-1/PD-L1 axis, which naturally

limit T-cell responses and maintain tolerance, have been particularly

successful (Chap. 73).

Vaccination This strategy promotes more efficient recognition of

tumor cells by the immune system, but the development of a therapeutic vaccine, as opposed to the preventative vaccines required

to combat infectious diseases, has proved to be a considerable challenge. Approaches have included direct injection of tumor- or tumor

antigen–derived RNA or DNA; transduction of tumor cells with

immune-enhancing genes encoding cytokines, chemokines, or costimulatory molecules; and the ex vivo manipulation of dendritic cells

to enhance the presentation of tumor antigens. A dendritic cell vaccine

for treatment of recurrent prostate cancer has received approval in the

United States, but its limited potency and high cost have constrained

commercial success.

Adoptive Cell Transfer Host immune cells such as T cells, NK

cells, and others can be modified to express new transgenic receptors intended to recognize tumor cells and their microenvironment

(Fig. 470-1). Retargeting may use a modification of the cells’ own receptor or a molecularly synthesized chimeric antigen receptor (CAR) that

is usually composed of the antigen recognition portion of an antibody

and the signaling components of the cell’s native antigen receptor along

with one or more additional signaling domains that boost T-cell activation. Both approaches have been successful, with significant responses

reported with native receptors targeted to melanoma and synovial

cell sarcoma and—most dramatically—with CARs targeted to CD19,

an antigen expressed at high levels on normal and many malignant

B cells, or B-cell maturation antigen (BCMA), an antigen expressed

at high levels in normal and multiple myeloma plasma cells. Infused

CAR T cells can expand many thousand fold in vivo, persist long term,

and have produced >80% complete response rates when targeting

intractable B-cell acute lymphoblastic leukemia; approximately half of

treated patients have remained in remission for many years afterward

without further cancer therapies. This approach has also been successful in adult patients with relapsed or chemotherapy-refractory B

cell–derived large-cell lymphoma, mantle cell lymphoma, and multiple

myeloma. Many responses are sustained long term, and several of these

CAR T-cell products have been approved by the U.S. Food and Drug

Administration (FDA), as well as be international regulatory agencies,

and adopted as standard of care. The general approach of CAR T cells

is under rapid development, including trials with CAR T cells targeting

different antigens for solid tumors and other hematologic malignancies

and CAR T cells with different molecular structures and different gene

transfer vectors. Remaining challenges in the field and application of

adoptive T-cell approaches include (1) the immune inhibitory microenvironment associated with most solid tumors; recent studies further

modify the T cells with countermeasures to tumor inhibitory signals;

(2) acute and severe (though rarely fatal) systemic inflammatory and

neurologic toxicities during the phase of T-cell expansion and tumor

killing, which typically require access to intensive care for clinical

management; (3) the off-target or on-target but off-tumor effects that

may damage normal host tissues (e.g., normal B cells following CD19

CAR therapy); and (4) the cost, time, and complexity of manufacture,

which is a particular problem when antigens unique to each tumor’s

individual mutations are targeted (neoantigens), rather than widely

shared tumor-associated antigens.

Nonimmunologic Modifications to Host Gene transfer can be

used to protect normal cells from the toxicities of chemotherapy and

thereby increase the therapeutic index of these drugs. The most extensively

studied approach has been to transduce hematopoietic cells with genes

encoding resistance to chemotherapeutic agents, including the multidrug

resistance gene MDRI or the gene encoding O6

-methylguanine DNA

methyltransferase (MGMT). Although such approaches reduce hematologic toxicity, cytotoxic dose escalation quickly reveals dose-limiting

toxicities to other organ systems. Chemotherapy resistance can also be

engineered into immune cells redirected to target cancer to enable combination treatments with cells and chemotherapy.

Finally, gene transfer can be used to inhibit the host angiogenesis

required for tumor support, for example, by constitutive expression of

inhibitors such as angiostatin and endostatin or the transfer of T cells

genetically modified to recognize antigens specific to newly forming

vasculature. These studies are in the early phase.

■ COMBINATION APPROACHES—MODIFICATION

OF HOST AND TUMOR BY VIROTHERAPY

Immuno-oncolytic Viruses These viruses are genetically modified to replicate in malignant but not normal cells. The replicating

CH2 VH

CL

CH

CAR T cells Native T cells

T cells

Antigenic

peptide

TCR complex

CAR

Spacer

CD3ζ

COSTIM

TM

TCR complex

γ ε ε δ ζ

γ ε ε δ ζ ζ

Monoclonal

antibody

Target cell TCR

β2

β

β

α

α

MHC I

VL

CH3

VL

VH

ζ ζ

FIGURE 470-1 T-cell receptors. A native T-cell receptor (TCR) recognizes processed

peptide antigens bound to major histocompatibility (MHC) molecules through its

αβ chains. Signaling then occurs through a multichain intracellular CD3 complex.

A chimeric antigen receptor (CAR) usually contains an extracellular receptor

component derived from the antigen binding portion (VH and VL

) of a monoclonal

antibody. This produces a receptor that can recognize either protein or nonprotein

antigens independent of the MHC. A transmembrane (TM) domain then connects

this receptor to the ζ chain of the CD3 complex derived from the native TCR. A

costimulation domain (COSTIM), such as CD28 or 4-1BB, is also present.

3690 PART 16 Genes, the Environment, and Disease

TABLE 470-5 Taking History from Subjects Enrolled in Gene Transfer

Studies

Elements of History for Subjects Enrolled in Gene Transfer Trials

1. What vector was administered? Is it predominantly integrating (retroviral,

lentiviral, herpesvirus [latency and reactivation]) or nonintegrating (plasmid,

adenoviral, adeno-associated viral)?

2. What was the route of administration of the vector?

3. What was the target tissue?

4. What gene was transferred in? A disease-related gene? A marker?

5. Were there any adverse events noted after gene transfer?

Screening Questions for Long-Term Follow-Up in Gene Transfer

Subjectsa

1. Has a new malignancy been diagnosed?

2. Has a new neurologic/ophthalmologic disorder, or exacerbation of a

preexisting disorder, been diagnosed?

3. Has a new autoimmune or rheumatologic disorder been diagnosed?

4. Has a new hematologic disorder been diagnosed?

a

Factors influencing long-term risk include integration of the vector into the

genome, vector persistence without integration, and transgene-specific effects.

“All disease begins in the gut.”

—Hippocrates

Nearly two and a half millennia after Hippocrates made this statement,

we are just coming to truly appreciate its profundity. Since the beginning of humankind, scholars have been investigating the underpinnings of disease with an almost singular focus on the human side of

the equation. Microbes were not recognized as an important cause of

disease until the inception of the “germ theory” in the late nineteenth

century. During the first century of medical microbiology, research

largely centered on the role of microbes as pathogens. Only recently has

there been a resurgence of interest in understanding how commensal

organisms—the bacteria, viruses, fungi, and Archaea that make up

the microbiota—impact human physiology. The idea that these microorganisms are vital to the well-being of humans has challenged our

traditional notions of “self.” Indeed, a human being can most accurately

be described as a holobiont: a complex assemblage of human cells and

microorganisms interacting in an elaborate pas de deux that drives

normal physiologic processes.

Aimed at a better understanding of this relationship, myriad studies during the past decade have begun to catalogue the microbiota at

various body sites and in a multitude of disease conditions. Diseases in

virtually every organ system have been associated with changes in the

microbiota. Indeed, the microbiota has been linked to intestinal disorders, disturbances in metabolic function, autoimmune diseases, and

psychiatric conditions and has been shown to influence susceptibility

to infection and the efficacy of pharmaceutical therapies. Knowledge

of the specific mechanism(s) underlying most of these microbe–disease

associations is lacking; it remains unclear whether the disease-associated

alterations in the microbiota represent mere biomarkers of disease, a

471 The Human Microbiome

Neeraj K. Surana, Dennis L. Kasper

vectors thus proliferate and spread within the tumor, facilitating eventual tumor clearance. However, physical limitations to viral spread,

including fibrosis, intermixed normal cells, basement membranes,

and necrotic areas within the tumor, may reduce clinical efficacy, and

their activity against metastatic disease has proved limited. Recently,

the FDA granted licensing approval to talimogene laherparepvec, an

oncolytic herpes virus containing the human granulocyte-macrophage

colony-stimulating factor gene, for treatment of melanoma. This

success has led to resurgent interest in combining the local tumor

destruction and tumor antigen release mediated directly by oncolytic

viruses with the recruitment of a systemic immune response mediated

by immunostimulatory genes contained within the oncolytic virus. In

principle, such immune-oncolytic viruses should produce responses in

both local and metastatic disease. Numerous novel viral agents are now

entering early-phase clinical testing.

■ OTHER APPROACHES

This chapter has focused on gene addition therapy, in which a normal

gene is transferred to a target tissue to drive expression of a gene product with therapeutic effects. Another powerful technique beginning

to yield dramatic clinical results is genome editing, which has the

potential to correct a mutation in situ, generating a wild-type copy

under the control of the endogenous regulatory signals. This approach

makes use of novel reagents including zinc finger nucleases, TALENs,

and CRISPR, which introduce breaks into the DNA near the site of the

mutation and then rely on a donated repair sequence and cellular mechanisms for repair to reconstitute a functioning gene. Another strategy

recently introduced into clinical trials is the use of small interfering

RNAs or short hairpin RNAs as transgenes to knock down expression

of deleterious genes (e.g., mutant huntingtin in Huntington’s disease).

SUMMARY

The power and versatility of gene therapy approaches are such that

there are few serious disease entities for which gene therapies are

not under development. The development of new classes of therapeutics typically takes two to three decades; monoclonal antibodies

and recombinant proteins are recent examples. Gene therapeutics,

which entered clinical testing in the early 1990s, traversed the same

time course. Examples of clinical success are now abundant, and gene

therapy approaches are likely to become increasingly important as a

therapeutic modality in the twenty-first century. A central question to

be addressed is the long-term safety of gene transfer, and regulatory

agencies have mandated a 15-year follow-up for subjects enrolled in

gene therapy trials (Table 470-5). Realization of the therapeutic benefits of modern molecular medicine will depend on continued progress

in gene transfer technology.

■ FURTHER READING

Al-Zaidy SA et al: AVXS-101 (onasemnogene abeparvovec) for

SMA1: Comparative study with a prospective natural history cohort.

J Neuromusc Dis 6:307, 2019.

Fesnak AD et al: Engineered T cells: The promise and challenges of

cancer immunotherapy. Nat Rev Cancer 16:566, 2016.

Fischer A, Hacein-Bey-Abina S: Gene therapy for severe combined

immunodeficiencies and beyond. J Exp Med 217:e20190607, 2020.

Frangoul H et al: CRISPR-Cas9 gene editing for sickle cell disease

and b-thalassemia. N Engl J Med 384:252, 2021.

High KA, Roncarolo MG: Gene therapy. N Engl J Med 381:455,

2019.

Hocquemiller M et al: Adeno-associated virus-based gene therapy

for CNS diseases. Hum Gene Ther 27:478, 2016.

June CH, Sadelain M: Chimeric antigen receptor therapy. N Engl J

Med 379:64, 2018.

Ramos CA et al: CAR-T cell therapy for lymphoma. Annu Rev Med

67:165, 2016.

Russell S et al: Efficacy and safety of voretigene neparvovec (AAV2-

hRPE65v2) in subjects with RPE65-mediated inherited retinal dystrophy: A randomised, controlled, open label phase 3 trial. Lancet

390:849, 2017.

Sessa M et al: Lentiviral haemopoietic stem-cell gene therapy in

early-onset metachromatic leukodystrophy: An ad-hoc analysis of a

non-randomised, open-label, phase 1/2 trial. Lancet 388:476, 2016.

Thompson AA et al: Gene therapy in patients with transfusiondependent β-thalassemia. N Engl J Med 378:1479, 2018.

The Human Microbiome

3691CHAPTER 471

diseases. Thus, although the field of microbiome research is sometimes

considered to have emerged over the last one or two decades, the basic

tenets—that the microbiota varies according to body site and clinical

characteristics, that microbes are critical for human health, and that

specific modulation of the microbiota may lead to improved clinical

outcomes—are far from new.

A PRIMER ON TAXONOMY

Given that microbiome-based studies have identified and compared

microbes at different levels of taxonomic resolution (Fig. 471-1), some

understanding of taxonomy is essential for better comprehension of

the implications of these studies. Of the ~100 bacterial phyla that

exist in nature, only five (Actinobacteria, Bacteroidetes, Firmicutes,

Fusobacteria, and Proteobacteria) are dominant members of the

human microbiome. Each of these phyla can be further categorized

into multiple classes, orders, families, genera, and species. Early studies

on the microbiota focused on changes in the relative abundance at the

phylum level between different groups (e.g., obese vs normal-weight

patients); however, these comparisons are at such a broad taxonomic

level that they often provide little or no biologic insight. As illustrated

in Fig. 471-1, drawing comparisons between organisms in two different bacterial phyla is analogous to comparing humans to sea stars: the

evolutionary distance between the two is tremendous. The limitations

of current bioinformatic tools require lumping together of taxonomically related strains and thus cloud the richness of microbial ecology.

Examining microbial profiles at the phylum, family, or even genus

level—as is often done at present—ignores the great heterogeneity

within different strains of the same bacterial species. The analytical

pipelines are just now beginning to enable strain-level comparisons,

and these improvements will likely facilitate our ongoing investigation

of host–commensal interactions.

THE MICROBIOTA AND HUMAN HEALTH

■ OVERVIEW OF THE HUMAN MICROBIOTA

The overwhelming majority of microbiota studies have focused on

stool, given that this sample type represents the most ecologically rich

anatomic site, is easy to obtain, and can readily be followed longitudinally in the same individual. A landmark study by the HMP sought to

define the “normal” microbiota throughout the entire body in healthy

Western adults. To this end, the microbial populations at 15–18 body

sites were characterized in 242 people. One striking finding was that

all samples from a given body region (e.g., skin) were more similar

to each other than they were to samples from a different body region

(e.g., stool), even in the same individual (Fig. 471-2A). In essence, the

effect of the anatomic site on microbial composition is far greater than

the effect of heterogeneity between individuals. That said, there was

a remarkable amount of interindividual variation at any given body

site (Fig. 471-2B). In stool, for example, the abundance of the phylum

Domain

Phylum

Kingdom

Class

Order

Family

Genus

Species

Staphylococcus

Staphylococcaceae

Bacillales

Bacilli

Firmicutes

Bacteria

Primate

Hominidae

Homo

Mammalia

Chordate

Animalia

Eukarya

S. aureus

S. epidermidis

S. lugdunensis

G. haemolysans

M. equipercicus

L. monocytogenes

B. anthracis

S. pneumoniae

E. faecalis

C. botulinum

C. difficile

E. rhusiopathiae

E. coli

B. fragilis

FIGURE 471-1 Juxtaposition of bacterial and human taxonomy highlights the evolutionary distance between different taxonomic levels. The listed species represent

exemplars that are members of the taxon to which they are connected but that are not contained within the next-lower-level taxon listed. For example, Clostridium botulinum,

Clostridium difficile, and Erysipelothrix rhusiopathiae are members of the phylum Firmicutes, but are in classes other than Bacilli. Similarly, starfish and humans are both

members of the kingdom Animalia, but they are in different phyla.

causal relationship, or a combination of the two. Although cause-andeffect relationships are still being elucidated for many diseases, it is

clear that humans coexist in an intricate relationship with commensal

organisms. This chapter explores in detail the nature of these host–

commensal interactions, focusing on how this information might be

translated into clinically meaningful interventions.

HISTORICAL PERSPECTIVE

Massive undertakings, such as the Human Microbiome Project (HMP)

sponsored by the National Institutes of Health and MetaHIT sponsored

by the European Commission, have catalogued all the bacteria present

at multiple body sites in people with and without disease. Coupled with

the confluence of advances in sequencing technologies (Chap. 121),

gnotobiotic animal availability, and microbial culture, significant progress has been made toward an understanding of the interplay between

the microbiota and human health. However, recent findings were foreshadowed by work done centuries ago.

The human microbiota was first explored in 1683 when Antony

van Leeuwenhoek described in a letter to the Royal Society of London

the “very little living animalcules, very prettily a-moving” that he had

observed in the plaque between his teeth. Leeuwenhoek went on to

perform the first comparative “microbiota” studies by assessing how

fecal and oral bacteria differ, how oral microbes change in the setting

of disease (e.g., alcoholism and tobacco use), and how microbial composition changes across the age spectrum (e.g., in young children vs

old men). He attempted—unsuccessfully—to eliminate these bacteria.

Although Leeuwenhoek was not taken seriously when he first reported

his findings, his studies laid the groundwork for what is now the field

of microbiome research, and investigators are still trying to answer

many of the same overarching questions that he raised more than three

centuries ago.

Although Leeuwenhoek first reported the existence of bacteria and

their association with humans at the end of the seventeenth century,

the significance of commensal bacteria was not realized until late in

the nineteenth century. In 1885, Pasteur suggested that animals could

not survive if they were “artificially and completely deprived of the

common microbes.” Although Pasteur’s preconceived ideas were proven

incorrect in 1912 by the advent of germ-free animals (animals raised

without exposure to any microorganisms), the underlying concept that

commensal organisms are critical to health has held up. Élie Metchnikoff

made another conceptual advance in this field by suggesting at the

beginning of the twentieth century that clinical outcomes could be

altered by the administration of specific beneficial organisms (probiotics). In particular, Metchnikoff believed that aging was caused by

toxic bacteria in the gut and that lactic acid–producing bacteria (e.g.,

Lactobacillus species) present in sour milk and yogurt could mitigate

against this process. The data behind this specific claim are still lacking, but recent discoveries offer continued hope that the microbiome

can be effectively harnessed to protect against and treat a variety of

3692 PART 16 Genes, the Environment, and Disease

Bacteroidetes ranged from ~10% in some individuals to >90% in others. Remarkably, even with person-to-person variability and differences

among body sites, the functional capacity of the microbiota—assessed

using metagenomic data to identify gene pathways—was quite similar

across different people and different body sites (Fig. 471-2C). This discrepancy between the substantial differences in microbial composition

and the little or no resulting change in the functional properties of the

microbiota reflects an important ecologic property of the microbiota:

the microbial communities at different body sites and in different

people assemble in such a way that all the core metabolic functions

are maintained. This finding also hints at the likely possibility of significant functional redundancy within the microbiota, with different

species executing the same biologic functions in different people and/

or at different anatomic sites.

While the HMP provided the first large-scale catalogue of the microbiome in multiple people and at many different body sites, the amount

of data generated by what, at the time, was by far the largest microbiome study has been dwarfed by subsequent studies. These more recent

studies have confirmed the HMP’s major tenets: the composition of

the microbiota differs by body site, there is tremendous interindividual

variation, and the microbial gene content is relatively conserved irrespective of the body site or individual. No microbial species are ubiquitous in all individuals and at all body sites, but some species are highly

prevalent at a given body site: in the HMP study, Staphylococcus epidermidis was present in 93% of nares samples and Escherichia coli in 61%

of stool samples. These findings highlight the remarkable personalization of the human microbiome. While the human genome is typically

>99.5% identical in different people, the microbiotas of two individuals

may not overlap at all. Although the “precision medicine” approach

currently focuses on teasing out how differences in the human genome

relate to different clinical end points, the human microbiome clearly

represents a critical component for consideration.

■ THE MICROBIOTA BY THE NUMBERS

It has long been known that the human-associated microbiota is numerically dense. Leeuwenhoek estimated that there were more “animals

living in the scrum on the teeth in man’s mouth than there are men in

a kingdom.” Specific enumeration of the components of the microbiota

has been challenging, in part because of its variability across time,

space (body region), and clinical conditions. Moreover, the majority

of human-associated microbes are not readily cultivable—a situation

that raises questions about the best methodology for such quantitation.

Initial back-of-the-envelope calculations performed in the 1970s suggested that there were roughly tenfold more bacteria in the body than

there were human cells. This rather astounding estimate suggested

that humans are really only ~10% “human” and that by far the greatest

part of the holobiont is represented by microbes. This stark numerical

discrepancy has prompted some to question “who parasitizes whom.”

However, a more recent estimate has suggested that there are “only”

~1.3 times more bacteria in the body than there are human cells and

thus that humans are ~56% “bacterial.” Of note, this more recent

study does not include the numbers of viruses (known to generally

be approximately tenfold more abundant than other microbes), fungi,

or Archaea. Given these additional microorganisms, the notion that

microbes constitute >90% of the cells present in a human body is

likely correct. These ratios are even starker when one considers the

genetic potential of human cells versus that of commensal organisms.

In contrast to the ~20,000 genes in the human genome, the estimated

Phyla

Firmicutes

Actinobacteria

Bacteroidetes

Proteobacteria

Fusobacteria

Tenericutes

Spirochaetes

Cyanobacteria

Verrucomicrobia

TM7

Metabolic pathways

Central carbohydrate metabolism

Cofactor and vitamin biosynthesis

Oligosaccharide and polyol transport system

Purine metabolism

ATP synthesis

Phosphate and amino acid transport system

Aminoacyl tRNA

Pyrimidine metabolism

Ribosome

Aromatic amino acid metabolism

B

C

Anterior nares RC Buccal mucosa Supragingival plaque Tongue dorsum Stool Posterior fornix PC2 (4.4%)

PC1 (13%)

Gastrointestinal

Urogenital

Skin

Nasal

Oral

A

FIGURE 471-2 The human microbiome exhibits significant taxonomic variability among body sites and between individuals while maintaining core metabolic pathways.

A. Principal coordinates (PC) plot showing variation among samples demonstrates that primary clustering is by body area, with the oral, gastrointestinal, skin, and urogenital

habitats separate; the nares habitat bridges oral and skin habitats. Each circle represents an individual sample. B, C. Vertical bars represent microbiome samples by

body habitat, with each bar within a given body site representing a different individual. Bars indicate relative abundances colored by microbial phyla (B) and metabolic

pathways (C). The legend on the right indicates the most abundant phyla/pathways. RC, retroauricular crease. (Reproduced with permission from Human Microbiome

Project Consortium: Structure, function and diversity of the healthy human microbiome. Nature 486:207, 2012.)

The Human Microbiome

3693CHAPTER 471

Supragingival plaque

GI/Stool

Skin

Vagina

Actinobacteria

Bacteroidetes

Fusobacteria

Proteobacteria

Firmicutes

Other

KEY

Buccal mucosa

Nares

FIGURE 471-3 Different anatomic sites harbor very different microbiomes. The figure indicates the relative

proportion of sequences determined at the taxonomic phylum level at six anatomic sites. (Data for stool, vagina,

nares, buccal mucosa, and supragingival plaque are from the Human Microbiome Project; data for the skin are

from EA Grice et al: Topographical and temporal diversity of the human skin microbiome. Science 324:1190,

2009.)

total number of genes in the microbiota (which

together constitute the microbiome)—i.e.,

>2,000,000—indicates that the human genome

contributes <1% to the total genetic potential

of the overall holobiont. Most microbiome

studies to date have focused almost exclusively

on the bacterial component; much remains to

be learned about the functional interplay of

bacteria, viruses, fungi, and Archaea and how

these other classes of microorganisms impact

human health.

In terms of overall diversity, >10,000 different bacterial species are present in the human

microbiota; the intestines alone contain >1000

species. At any given time, the body of any given

individual harbors 500–1000 bacterial species,

with 100–200 bacterial species in the gut alone.

If one considers different strains of the same

bacterial species, which may be functionally

different from one another, the diversity of the

microbiota is probably at least a magnitude

greater. Although marked diversity exists at the

strain and species level, only limited bacterial

phyla are generally found in the human microbiota at any given body site (Fig. 471-3).

■ INFLUENCES ON THE

MICROBIOTA

An individual’s specific microbial configuration

is dynamic and is quickly altered in response

to subtle changes in the microenvironments

in which the bacteria reside. On a day-to-day

basis, these changes usually reflect alterations in

the relative abundance of the various microbes.

However, some exposures have a greater effect

on the microbiota and can shift the microbial

population to a new equilibrium via the loss

of specific species and/or the acquisition of

others; this new microbial equilibrium can be

associated with either health or a disease state

(Fig. 471-4). Identification of the factors that influence the microbiota’s composition is critical to an understanding of what leads to and

controls intra- and interindividual variation. Moreover, an understanding of the influences on the microbiota will facilitate the design and

proper interpretation of microbiota studies. While it is clear that the

microbiota can be altered through these various mechanisms, it is not

yet clear whether these changes are biologically significant.

Genetics Studies of monozygotic and dizygotic twins have revealed

that host genetics have a small but statistically significant effect on the

microbiota’s composition. Notably, some taxa, such as Christensenella

species, are more heritable than others. A cross-sectional study of

>1000 healthy individuals who have distinct ancestral origins and a

relatively shared common environment confirmed a weak association

between host genetics and the microbiome but highlighted that environmental factors are more prominent modulators of the microbiome.

That said, the host’s genetic contribution to the microbiota, albeit

small, may be meaningful. Studies in mice have demonstrated that

genetic variation in the major histocompatibility complex, a specific set

of immune-related genes, leads to changes in the microbiota that alter

susceptibility to an autoimmune disease. These studies offer a proof

of concept for the notion that the genetic predisposition observed for

certain diseases may actually be mediated by indirect alterations in the

microbiota.

Age Burgeoning evidence now indicates that microbial exposure

may begin in utero: bacterial DNA from bacteria typically associated

with the oral microbiota has been identified in otherwise healthy

placentas, in amniotic fluid obtained at early stages of gestation, and

in meconium of term newborns. Although some controversy persists

about whether these results reflect contamination and/or the presence

of nonviable bacteria, they raise the possibility that human exposure to

the microbial world begins before birth. The delivery mode (vaginal vs

cesarean section) and the method of feeding (breast milk vs formula,

timing of solid food introduction) are major determinants of an infant’s

Unstable

Stable

Healthy state 1

Current microbial

state

Disease state

Healthy state 2

FIGURE 471-4 A stability landscape of the human microbial ecosystem. A stable

state, illustrated as a depression in the landscape, can be associated with either a

healthy state or a disease state. The topology of an individual’s landscape reflects

that person’s genetics, age, diet, medications, medical history, and lifestyle. The

position of the green ball represents the current microbial state. Clinical changes

(e.g., administration of antibiotics, development of disease) can influence both the

current state and the overall topology.

3694 PART 16 Genes, the Environment, and Disease

1

D Strachan: BMJ 299:1259, 1989.

early microbiota. After birth, the infant’s microbiota goes through a

stereotyped succession process; with increases in bacterial diversity

and functional capacity, the child’s microbiota resembles that of an

adult by the age of 2–3 years. Cross-sectional studies that have examined the microbiota across the entire age spectrum have revealed a

general stability of the fecal microbiota after 2–3 years of age; however,

the microbiota of the elderly (persons >80 years of age) demonstrates

notable differences from those of their younger counterparts, with

increases in Bacteroides and Eubacterium species and decreases in the

bacterial family Lachnospiraceae.

Diet Diet is a strong determinant of human health. The impact of

diet is mediated, in part, by its effects on the composition of the gut

microbiota. This makes intuitive sense, as the human diet provides

nutrients needed not only by our own cells but also by the microbes

living in the alimentary tract. In young children, this dietary influence

is marked by major shifts (e.g., a decrease in Bifidobacterium species) in

the intestinal microbiota that occur at weaning and with the introduction of solid food. In adults, long-term dietary patterns are associated

with relatively stable microbial compositions. However, drastic changes

in short-term macronutrient availability cause rapid (within 1 day)

and reproducible fluctuations in the fecal microbiota that reflect the

biologic processes needed to degrade and metabolize the nutrients in

the new diet. For example, vegetarian diets are associated with a microbiota that has an increased ability to metabolize plant polysaccharides

(e.g., Roseburia species, Eubacterium rectale, Ruminococcus bromii),

while animal-based diets result in an increased abundance of biletolerant organisms (e.g., Alistipes, Bilophila, and Bacteroides species).

At the completion of dietary interventions and the resumption of the

individual’s normal dietary pattern, the microbial communities revert

back to their previous states, probably because the individual resumes

his or her typical diet. Taken together, dietary studies confirm that

the microbiota is highly adaptable and varies in relation to changes in

the diet. Of note, virtually all of these studies have focused on how the

diet influences the fecal microbiota. It will be interesting to determine

whether dietary changes similarly influence the microbiota at nonintestinal sites.

Drugs Virtually all drugs have the capacity to change the microbiota

by altering the chemical landscape in which the microorganisms live

(e.g., statins, bile acid sequestrants), modulating the host’s ability to

recognize and react to microbes (e.g., immunosuppressants) and/or

directly interfering with the microbiota’s constituents (e.g., antibiotics).

These potential effects have made critical interpretation of microbiota

studies much more difficult. A prominent study that claimed to identify a fecal microbiota signature associated with type 2 diabetes was

later found actually to have identified a signature for patients taking

metformin instead; the effects of this drug on the microbiota were far

greater than the effects of the disease itself. These results highlight the

importance of controlling for clinical variables in microbiota studies.

Antibiotics are the most obvious and best-studied class of drugs

that modulate the microbiota. Multiple groups have demonstrated

that antibiotics exert a considerable effect on the gut microbiota by

depleting antibiotic-sensitive strains. What is more surprising is that

many strains resistant to the antibiotic tested are also eliminated. This

observation highlights the intricate microbe–microbe interactions that

are fundamental to maintenance of the overall microbial community.

For example, treatment with ciprofloxacin, which has little to no activity against clinically relevant anaerobes, leads to a loss of roughly onethird of the bacterial taxa in the gut. This broad effect is likely mediated

by the depletion of certain “keystone” species that are required for the

persistence of other, unrelated species. While many of the observed

antibiotic effects (e.g., loss of specific taxa) are shared across many

different individuals, some effects vary greatly among people. For

example, studies found that microbiota recovery following antibiotic

treatment differed significantly in terms of timing and degree. The

microbiota of most healthy people who received ciprofloxacin for

5 days had completely recovered within 4 weeks, whereas microbiologic changes lasted up to 6 months in other individuals. Moreover,

the degree of variation was compounded by repeated antibiotic

administration, with fewer individuals reverting to their baseline

microbiota after a second course of ciprofloxacin given 6 months after

the first. These findings are consistent with those of microbial ecology

experiments, which also showed that this type of repeated disturbance

leads to less predictable results.

Lifestyle Many seemingly innocuous lifestyle decisions can impact

the human microbiota. For example, a person’s skin and fecal microbiotas are more similar to those of their household members, regardless of

genetic relatedness, than to those of residents of different households.

The degree of similarity in skin microbiotas is even greater if a dog also

lives in the home; in contrast, the presence of a young child does not

accentuate this microbial relatedness. The presumption is that the dog

serves as a more effective “vector” for transmitting microbes during

its frequent direct contact with adults in the household. The type of

setting in which a person lives also impacts the microbiota. Living in a

rural or farm setting leads to a different fecal microbiota than living in

an urban environment. Similarly, the individual’s country of residence

affects the microbiota. An analysis of daily fecal samples from an individual who temporarily (i.e., for a couple of months) moved from the

United States to Thailand demonstrated a large shift in the fecal microbiota that coincided with arrival in Thailand and a reversion in most

respects to the “American” microbial configuration upon return to the

United States. Similarly, immigration to the United States “westernizes”

the microbiome of individuals coming from non-Western countries.

These geography-driven changes probably reflect a combination of

environmental and dietary differences between locations.

Circadian Rhythms Many human biologic processes follow a circadian clock; aspects of physiology are tuned by external cues, including

the degree and timing of ambient light, temperature, and availability of

nutrients. This endogenous biologic clock enables animals to efficiently

adapt to changing environmental conditions. Similarly, the microbiota

maintains a circadian rhythm that is linked to—and helps entrain—the

host’s circadian clock. If circadian oscillations are disrupted in the host,

they are similarly disrupted in the microbiota, and vice versa. These

bacterial vacillations occur at the level of spatial localization within the

intestine, relative species abundance, and bacterial metabolite secretion. Work in the 1960s showed that mice exhibited daily periodicity

of susceptibility to infection with either Streptococcus pneumoniae or

E. coli lipopolysaccharide. Although the fundamental basis for this

difference was not known at the time, it is likely to be related, in part,

to the microbial circadian clock. Derangements of these microbial

oscillations have also been linked to the development of metabolic

diseases and may underlie some of the health hazards associated with

shift work and jet lag.

THE MICROBIOTA AND DISEASE

■ THE HYGIENE HYPOTHESIS

Over the past few decades, abundant epidemiologic data have revealed

an inverse correlation between exposure to microbes and the incidence

of autoimmune and/or atopic diseases (Fig. 471-5). This type of epidemiologic correlation led to the proposal of the “hygiene hypothesis”

in 1989. Initially, this hypothesis focused on the development of atopic

diseases in young children, with the idea that these epidemiologic

observations could “be explained if allergic diseases were prevented by

infection in early childhood, transmitted by unhygienic contact with

older siblings, or acquired prenatally from a mother infected by contact

with her older children.”1

 In fact, this notion that differences in living

conditions and environmental exposures contribute to susceptibility to

hay fever (summer catarrh) dates back to at least the early nineteenth

century. The hygiene hypothesis has continued to evolve over the past

three decades and now posits that inadequacies in microbial exposure—

in combination with genetic susceptibilities—lead to a collapse of the

normally highly coordinated, homeostatic immune response. At its

core, the hygiene hypothesis holds that specific early-life microbial